Personalised immunogenic peptide identification platform

ABSTRACT

The disclosure relates to methods of identifying fragments of a polypeptide that are immunogenic for a specific human subject, methods of preparing personalised pharmaceutical compositions comprising such polypeptide fragments, human subject-specific pharmaceutical compositions comprising such polypeptide fragments, and methods of treatment using such compositions. The methods comprise identifying a fragment of the polypeptide that binds to multiple HLA of the subject.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.15/910,930, filed Mar. 2, 2018, which claims the benefit of priority toEuropean Application No. 17159242.1, filed on Mar. 3, 2017, EuropeanApplication No. 17159243.9, filed on Mar. 3, 2017, and Great BritainApplication No. 1703809.2, filed on Mar. 9, 2017, each of which isincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created Sep. 14, 2021, isnamed “TBL_003C1_SL.txt” and is 25,720 bytes in size.

FIELD

The disclosure relates to methods of predicting whether a polypeptide isimmunogenic for a specific human subject, methods of identifyingfragments of a polypeptide that are immunogenic for a specific humansubject, methods of preparing personalised or precision pharmaceuticalcompositions or kits comprising such polypeptide fragments, humansubject-specific pharmaceutical compositions comprising such polypeptidefragments, and methods of treatment using such compositions.

BACKGROUND

For decades, scientists have assumed that chronic diseases were beyondthe reach of a person's natural defences. Recently, however, significanttumor regressions observed in individuals treated with antibodies thatblock immune inhibitory molecules have accelerated the field of cancerimmunotherapy. These clinical findings demonstrate that re-activation ofexisting T cell responses results in meaningful clinical benefit forindividuals. These advances have renewed enthusiasm for developingcancer vaccines that induce tumor specific T cell responses.

Despite the promise, current immunotherapy is effective only in afraction of individuals. In addition, most cancer vaccine trials havefailed to demonstrate statistically significant efficacy because of alow rate of tumor regression and antitumor T cell responses inindividuals. Similar failures were reported with therapeutic andpreventive vaccines that sought to include T cell responses in thefields of HIV and allergy. There is a need to overcome the clinicalfailures of immunotherapies and vaccines.

SUMMARY

In antigen presenting cells (APC) protein antigens are processed intopeptides. These peptides bind to human leukocyte antigen molecules(HLAs) and are presented on the cell surface as peptide-HLA complexes toT cells. Different individuals express different HLA molecules anddifferent HLA molecules present different peptides. Therefore, accordingto the state of the art, a peptide, or a fragment of a largerpolypeptide, is identified as immunogenic for a specific human subjectif it is presented by a HLA molecule that is expressed by the subject.In other words, the state of the art describes immunogenic peptides asHLA-restricted epitopes. However, HLA restricted epitopes induce T cellresponses in only a fraction of individuals who express the HLAmolecule. Peptides that activate a T cell response in one individual areinactive in others despite HLA allele matching. Therefore, it wasunknown how an individual's HLA molecules present the antigen-derivedepitopes that positively activate T cell responses.

As provided herein multiple HLA expressed by an individual need topresent the same peptide in order to trigger a T cell response.Therefore the fragments of a polypeptide antigen that are immunogenicfor a specific individual are those that can bind to multiple class I(activate cytotoxic T cells) or class II (activate helper T cells) HLAsexpressed by that individual.

Accordingly, in a first aspect the disclosure provides methods ofpredicting whether a polypeptide or a fragment of a polypeptide isimmunogenic for a specific human subject, the methods comprising thesteps of

-   -   (i) determining whether the polypeptide comprises:        -   (a) an amino acid sequence that is a T cell epitope capable            of binding to at least two HLA class I molecules of the            subject; or        -   (b) an amino acid sequence that is a T cell epitope capable            of binding to at least two HLA class II molecules of the            subject; and    -   (ii) predicting        -   A. that the polypeptide is immunogenic for the subject if            the polypeptide comprises at least one sequence that meets            the requirements of step (i); or        -   B. that the polypeptide is not immunogenic for the subject            if the polypeptide does not comprise at least one sequence            that meets the requirements of step (i).

The disclosure also provides methods of identifying a fragment of apolypeptide as immunogenic for a specific human subject, the methodscomprising the steps of

-   -   (i) determining that the polypeptide comprises:        -   (a) an amino acid sequence that is a T cell epitope capable            of binding to at least two HLA class I molecules of the            subject; or        -   (b) an amino acid sequence that is a T cell epitope capable            of binding to at least two HLA class II molecules of the            subject; and    -   (ii) identifying said sequence as a fragment of the polypeptide        that is immunogenic for the subject.

In some embodiments the methods of the disclosure comprise the step ofdetermining or obtaining the HLA class I genotype and/or the HLA classII genotype of the specific human subject.

A specific polypeptide antigen may comprise more than one fragment thatis a T cell epitope capable of binding to multiple HLA of a specificindividual. The combined group of all such fragments characterize theindividual's antigen specific T cell response set, wherein the aminoacid sequence of each fragment characterizes the specificity of eachactivated T cell clone.

Accordingly in some cases the method is repeated until all of thefragments of the polypeptide that are a T cell epitope capable ofbinding to at least two HLA class I and/or at least two HLA class II ofthe subject have been identified. This method characterises the immuneresponse of the subject to the polypeptide.

The disclosure further provides methods of treatment of a human subjectin need thereof, the method comprising administering to the subject apolypeptide, pharmaceutical composition or kit of the polypeptides of apanel of polypeptides that has been identified or selected by any of themethods above or comprising a fragment of a polypeptide that has beenidentified or selected by any of the methods above; their use in amethod of treatment of a relevant human subject; and their use in themanufacture of a medicament for treating a relevant subject.

The fragments of polypeptide that are determined to be immunogenic for aspecific human subject in accordance with the methods above can be usedto prepare human subject-specific immunogenic compositions.

Accordingly in a further aspect, the disclosure provides methods ofdesigning or preparing a human subject-specific pharmaceuticalcomposition or kit or panel of polypeptides for use in a method oftreatment of a specific human subject, the methods comprising:

-   -   (i) selecting a fragment of a polypeptide, which fragment has        been identified as immunogenic for the subject by the method        above;    -   (ii) if the fragment selected in step (i) is an HLA class        I-binding epitope, optionally selecting a longer fragment of the        polypeptide, which longer fragment        -   a. comprises the fragment selected in step (i); and        -   b. is a T cell epitope capable of binding at least three or            to the most possible HLA class II molecules of the subject;    -   (iii) selecting a first sequence of up to 50 consecutive amino        acids of the polypeptide, which consecutive amino acids comprise        the amino acid sequence of the fragment selected in step (i) or        the longer fragment selected in step (ii);    -   (iv) repeating steps (i) to (iii) to select a second amino acid        sequence of up to 50 consecutive amino acids of the same or a        different polypeptide to the first amino acid sequence;    -   (v) optionally further repeating steps (i) to (iii) to select        one or more additional amino acid sequences of up to 50        consecutive amino acids of the same or different polypeptides to        the first and second amino acid sequences; and    -   (vi) designing or preparing a subject-specific pharmaceutical        composition, kit or panel of polypeptides having as active        ingredients one or more polypeptides that together have all of        the amino acid sequences selected in the preceding steps,        optionally wherein one or more or each sequence is flanked at        the N and/or C terminus by additional amino acids that are not        part of the sequence of the polypeptides.

In some cases each peptide either consists of one of the selected aminoacid sequences, or consists of two or more of the amino acid sequencesarranged end to end or overlapping in a single peptide.

The disclosure further provides a human subject-specific pharmaceuticalcomposition, kit or panel of polypeptides for use in a method oftreatment of a specific human subject in need thereof, the composition,kit or panel comprising as active ingredients a first and a secondpeptide and optionally one of more additional peptides, wherein eachpeptide comprises an amino acid sequence that is a T cell epitopecapable of binding to at least two HLA class I molecules and/or at leasttwo HLA class II molecules of the subject, wherein the amino acidsequence of the T cell epitope of the first, second and optionally anyadditional peptides are different from each other, and wherein thepharmaceutical composition or kit optionally comprises at least onepharmaceutically acceptable diluent, carrier, or preservative.

The disclosure further provides a human subject-specific pharmaceuticalcomposition, kit or panel of polypeptides for use in a method oftreatment of a specific human subject in need thereof, the compositionor kit comprising as an active ingredient a polypeptide comprising afirst region and a second region and optionally one of more additionalregions, wherein each region comprises an amino acid sequence that is aT cell epitope capable of binding to at least two HLA class I moleculesand/or at least two HLA class II molecules of the subject, wherein theamino acid sequence of the T cell epitope of the first, second andoptionally any additional regions are different from each other, andwherein the pharmaceutical composition or kit optionally comprises atleast one pharmaceutically acceptable diluent, carrier, or preservative.

The disclosure further provides a method of designing or preparing apolypeptide for inducing an immune response in a specific human subjectthe method comprising selecting an amino acid sequence that is a T cellepitope capable of binding to at least three HLA class I molecules or atleast three HLA class II molecules of the subject, and designing orpreparing a polypeptide comprising the selected amino acid sequence.

In further aspects, the disclosure provides

-   -   a method of inducing an immune response or a method of treatment        comprising administering to a human subject in need thereof a        human subject-specific pharmaceutical composition, or the        polypeptides a kit or panel as described above, wherein the        composition, kit or panel of polypeptides is specific for the        subject;    -   a human subject-specific immunogenic composition, kit or panel        as described above for use in a method of inducing an immune        response or a method of treatment of the specific human subject;        and    -   use of a human subject-specific pharmaceutical composition or        the polypeptides of a kit or panel as described above in the        manufacture of a medicament, wherein the medicament is for        inducing an immune response in or treating the specific subject.

In a further aspect the disclosure provides a system comprising

(a) a storage module configured to store data comprising the class Iand/or class II HLA genotype of a subject and the amino acid sequence ofone or more test polypeptides; and(b) a computation module configured to identify and/or quantify aminoacid sequences in the one or more test polypeptides that are capable ofbinding to multiple HLA class I molecules of the subject and/or that areare capable of binding to multiple HLA class II molecules of thesubject.

The disclosure provides a method of treatment of a human subject in needthereof, the method comprising administering to the subject apolypeptide, a panel of polypeptides, a pharmaceutical composition orthe active ingredient polypeptides of a kit described above, wherein thesubject has been determined to express at least three HLA class Imolecules and/or at least three HLA class II molecules capable ofbinding to the polypeptide or to one or more of the active ingredientpolypeptides of the pharmaceutical composition or kit.

Disclosed herein in certain embodiments are human subject-specificpharmaceutical compositions for treatment of a disease or disorder in aspecific human subject, comprising (a) at least two differentpolypeptides, each of the at least two different polypeptides being10-50 amino acids in length and comprising a T cell epitope that bindsat least three HLA class I molecules of the subject and/or at leastthree HLA class II molecules of the subject, and wherein the T cellepitope of each of the at least two polypeptides are different from eachother; and (b) a pharmaceutically-acceptable adjuvant. In someembodiments, the composition comprises at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least11, or at least 12 different polypeptides. In some embodiments, thecomposition comprises 3-40 different polypeptides. In some embodiments,the T cell epitope that binds at least three HLA class I molecules ofthe subject comprises 7 to 11 amino acids, and/or the T cell epitopethat binds at least three HLA class II molecules comprises 13 to 17amino acids. In some embodiments, the epitopes of the at least twodifferent polypeptides are from a single antigen. In some embodiments,the epitopes of the at least two different polypeptides are from two ormore different antigens. In some embodiments, the antigen is an antigenexpressed by a cancer cell, a neoantigen expressed by a cancer cell, acancer-associated antigen, a tumor-associated antigen, or an antigenexpressed by a target pathogenic organism, an antigen expressed by avirus, an antigen expressed by a bacterium, an antigen expressed by afungus, an antigen associated with an autoimmune disorder, or is anallergen. In some embodiments, the cancer cell is from the subject. Insome embodiments, the antigen is selected from the antigens listed inTables 2 to 7. In some embodiments, the at least two differentpolypeptides further comprise up to 10 amino acids flanking the T cellepitope that are part of a consecutive sequence flanking the epitope ina corresponding antigen. In some embodiments, the at least two differentpolypeptides further comprise up to 10 amino acids flanking the T cellepitope that are not part of a consecutive sequence flanking the epitopein a corresponding antigen. In some embodiments, two of the at least twopolypeptides are arranged end to end or overlapping in a joinedpolypeptide. In some embodiments, the composition comprises two or moredifferent joined polypeptides, wherein the two or more different joinedpolypeptides comprise different epitopes from each other. In someembodiments, the joined polypeptides have been screened to eliminatesubstantially all neoepitopes that span a junction between the twopolypeptides and that (i) corresponds to a fragment of a humanpolypeptide expressed in healthy cells of the subject; (ii) is a T cellepitope capable of binding to at least two HLA class I molecules of thesubject; or (iii) meets both requirements (i) and (ii). In someembodiments, the at least two polypeptides do not comprise any aminoacid sequences that (i) correspond to a fragment of a human polypeptideexpressed in healthy cells; or (ii) correspond to a fragment of a humanpolypeptide expressed in healthy cells and is a T cell epitope capableof binding to at least two HLA class I molecules of the subject. In someembodiments, the composition further comprises a pharmaceuticallyacceptable diluent, carrier, preservative, or combination thereof. Insome embodiments, the adjuvant is selected from the group consisting ofMontanide ISA-51, QS-21, GM-CSF, cyclophosamide, bacillusCalmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone,isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins(KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete),mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols,polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), andcombinations thereof.

Disclosed herein in certain embodiments are kits comprising, one or moreseparate containers each container comprising: (i) one or morepolypeptides being 10-50 amino acids in length comprising an amino acidsequence that is a T cell epitope that binds at least three HLA class Imolecules of the subject and/or at least three HLA class II molecules ofthe subject; and (ii) a pharmaceutically acceptable adjuvant, diluent,carrier, preservative, or combination thereof. In some embodiments, thekit comprises at least 2, at least 3, at least 4, at least 5, at least6, at least 7, at least 8, at least 9, at least 10, at least 11, or atleast 12 different polypeptides, wherein the amino acid sequence of theT cell epitope of each of the different polypeptides are different fromeach other. In some embodiments, the kit further comprises a packageinsert.

Disclosed herein in certain embodiments are human subject-specificpharmaceutical compositions comprising: a nucleic acid moleculeexpressing two or more polypeptides, each polypeptide being 10-50 aminoacids in length comprising a T cell epitope that binds at least threeHLA class I molecules of the subject and/or at least three HLA class IImolecules of the subject, wherein each of the two or more polypeptidescomprises a different T cell epitope, wherein the polypeptides do notcomprise amino acid sequences that are adjacent to each other in acorresponding antigen. In some embodiments, the nucleic acid moleculeexpresses at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, or at least 12 differentpolypeptides, each being 10-50 amino acids in length comprising an aminoacid sequence that is a T cell epitope that binds at least three HLAclass I molecules of the subject and/or at least three HLA class IImolecules of the subject, wherein the amino acid sequence of the T cellepitope of each of the different polypeptides are different from eachother.

Disclosed herein in certain embodiments are human subject-specificpharmaceutical compositions for treatment of a disease or disorder in aspecific human subject, comprising at least one different polypeptides,each of the at least one different polypeptides comprising at least afirst region and a second region, (i) the first region of 10-50 aminoacids in length comprising an amino acid sequence that is a T cellepitope that binds at least three HLA class I molecules of the subjectand/or at least three HLA class II molecules of the subject, (ii) thesecond region of 10-50 amino acids in length comprising an amino acidsequence that is a T cell epitope that binds at least three HLA class Imolecules of the subject and/or at least two HLA class II molecules ofthe subject, wherein the amino acid sequence of the T cell epitope ofeach of the first and second regions of each of the at least threedifferent polypeptides comprise different sequences. In someembodiments, the composition comprises at least 2, at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, or at least 12 different polypeptides. In someembodiments, the composition comprises 2-40 different polypeptides. Insome embodiments, the T cell epitope that binds at least three HLA classI molecules of the subject comprises 7 to 11 amino acids, and/or the Tcell epitope that binds at least three HLA class II molecules comprises13 to 17 amino acids. In some embodiments, the epitopes of the first andsecond regions are from a single antigen. In some embodiments, theepitopes of the first and second regions are from two or more differentantigens. In some embodiments, the antigen is an antigen expressed by acancer cell, a neoantigen expressed by a cancer cell, acancer-associated antigen, a tumor-associated antigen, or an antigenexpressed by a target pathogenic organism, an antigen expressed by avirus, an antigen expressed by a bacterium, an antigen expressed by afungus, an antigen associated with an autoimmune disorder, or is anallergen. In some embodiments, the cancer cell is from the subject. Insome embodiments, the antigen is selected from the antigens listed inTables 2 to 7. In some embodiments, the polypeptides have been screenedto eliminate substantially all neoepitopes that span a junction betweenthe two regions and that (i) corresponds to a fragment of a humanpolypeptide expressed in healthy cells of the subject; (ii) is a T cellepitope capable of binding to at least two HLA class I molecules of thesubject; or (iii) meets both requirements (i) and (ii). In someembodiments, the at least one polypeptides do not comprise any aminoacid sequences that (i) correspond to a fragment of a human polypeptideexpressed in healthy cells; or (ii) correspond to a fragment of a humanpolypeptide expressed in healthy cells and is a T cell epitope capableof binding to at least two HLA class I molecules of the subject. In someembodiments, the composition further comprises a pharmaceuticallyacceptable adjuvant, diluent, carrier, preservative, or combinationthereof. In some embodiments, the adjuvant is selected from the groupconsisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, bacillusCalmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone,isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins(KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete),mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols,polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), andcombinations thereof.

Disclosed herein in certain embodiments are methods of preparing a humansubject-specific pharmaceutical composition for use in a method oftreatment of a specific human subject, the method comprising:

-   -   (i) selecting a fragment of a polypeptide, which fragment has        been identified as immunogenic for the subject by        -   a) determining whether the fragment comprises:            -   1) an amino acid sequence that is a T cell epitope                capable of binding to at least three HLA class I                molecules of the subject; or            -   2) an amino acid sequence that is a T cell epitope                capable of binding to at least three HLA class II                molecules of the subject; or            -   3) or meets both requirements (1) and (2); and        -   b) identifying the sequence as a fragment of the polypeptide            that is immunogenic for the subject;    -   (ii) selecting a first sequence of up to 50 consecutive amino        acids of the polypeptide, which consecutive amino acids comprise        the amino acid sequence of the fragment selected in step (i);        and    -   (iii) preparing a subject-specific pharmaceutical composition        having as active ingredients one or more polypeptides that        together have all of the amino acid sequences selected in the        preceding steps.

In some embodiments, the method further comprises prior to the preparingstep repeating steps (i) to (ii) to select a second amino acid sequenceof up to 50 consecutive amino acids of the same or a differentpolypeptide to the first amino acid sequence. In some embodiments, themethod further comprises, further repeating prior to the preparing step,steps (i) to (ii) one or more times to select one or more additionalamino acid sequences of up to 50 consecutive amino acids of the same ordifferent polypeptides to the first and second amino acid sequences. Insome embodiments, the method further comprises prior to the preparingstep selecting a longer fragment of the polypeptide if the fragmentselected in step (i) is an HLA class I-binding epitope, which longerfragment comprises the fragment selected in step (i); and is a T cellepitope capable of binding at least three HLA class II molecules of thesubject. In some embodiments, each polypeptide either consists of one ofthe selected amino acid sequences, or comprises or consists of two ormore of the selected amino acid sequences arranged end to end oroverlapping in a single joined polypeptide. In some embodiments, anyneoepitopes formed at the junction between any two of the selected aminoacid sequences arranged end to end in a single joined polypeptide havebeen screened to eliminate substantially all polypeptides comprising aneoepitope amino acid sequence that (i) corresponds to a fragment of ahuman polypeptide expressed in healthy cells; (ii) is a T cell epitopecapable of binding to at least two HLA class I molecules of the subject;or (iii) meets both requirements (i) and (ii). In some embodiments, theone or more polypeptides have been screened to eliminate polypeptidescomprising an amino acid sequence that (i) corresponds to a fragment ofa human polypeptide expressed in healthy cells; or (ii) corresponds to afragment of a human polypeptide expressed in healthy cells and is a Tcell epitope capable of binding to at least two HLA class I molecules ofthe subject. In some embodiments, the method further comprisesdetermining HLA class I genotype and HLA class II genotype from abiological sample of the subject prior to step (i). In some embodiments,the biological sample is obtained from the subject. In some embodiments,the determining HLA class I genotype and HLA class II genotype isperformed by sequence based typing (SBT) methods. In some embodiments,the determining HLA class I genotype and HLA class II genotype isperformed by sequencing, next generation sequencing, sequence specificprimer (SSP) methods, or sequence specific oligonucleotide (SSO)methods. In some embodiments, the biological sample is blood, serum,plasma, saliva, buccal swab, urine, expiration, cell, or tissue. In someembodiments, the biological sample is saliva or a buccal swab.

Disclosed herein in certain embodiments are methods of treating a cancerin a specific human subject in need thereof comprising, administering toa specific human subject a pharmaceutical composition comprising atleast one polypeptide, each of the at least one polypeptide being 10-50amino acids in length comprising a first amino acid sequence that is a Tcell epitope that binds at least three HLA class I molecules of thesubject and/or at least three HLA class II molecules of the subject,wherein the T cell epitope of each of the at least one polypeptide isfrom an antigen that is specific for the cancer. In some embodiments,the composition comprises at least 2, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least11, or at least 12 different polypeptides, wherein the amino acidsequence of the T cell epitope of each of the different polypeptides aredifferent from each other, and are from one or more antigens that areexpressed by a cancer cell from the subject. In some embodiments, thecomposition comprises 2-40 different polypeptides. In some embodiments,the T cell epitope that binds at least three HLA class I molecules ofthe subject comprises 7 to 11 amino acids, and/or the T cell epitopethat binds at least three HLA class II molecules comprises 13 to 17amino acids. In some embodiments, the composition comprises at least twodifferent polypeptides and the epitopes of the amino acid sequences ofthe at least two different polypeptides are from a single antigen. Insome embodiments, the composition comprises at least two differentpolypeptides and the epitopes of the at least two different polypeptidesare from two or more different antigens. In some embodiments, the one ormore antigen is a neoantigen expressed by a cancer cell, acancer-associated antigen, or a tumor-associated antigen. In someembodiments, the one or more antigen is selected from the antigenslisted in Table 2. In some embodiments, the at least one differentpolypeptides further comprise up to 10 amino acids flanking the T cellepitope that are part of a consecutive sequence flanking the epitope ina corresponding antigen. In some embodiments, the at least one differentpolypeptides further comprise up to 10 amino acids flanking the T cellepitope that are not part of a consecutive sequence flanking the epitopein a corresponding antigen. In some embodiments, the compositioncomprises at least two different polypeptides and two of thepolypeptides are arranged end to end or overlapping in a joinedpolypeptide. In some embodiments, the composition comprises two or moredifferent joined polypeptides, wherein the two or more different joinedpolypeptides comprise different epitopes from each other. In someembodiments, the joined polypeptides have been screened to eliminatesubstantially all neoepitopes that span a junction between the twopolypeptides and that (i) corresponds to a fragment of a humanpolypeptide expressed in healthy cells of the subject; (ii) is a T cellepitope capable of binding to at least two HLA class I molecules of thesubject; or (iii) meets both requirements (i) and (ii). In someembodiments, the at least one polypeptide does not comprise any aminoacid sequences that (i) correspond to a fragment of a human polypeptideexpressed in healthy cells; or (ii) correspond to a fragment of a humanpolypeptide expressed in healthy cells and is a T cell epitope capableof binding to at least two HLA class I molecules of the subject. In someembodiments, the composition further comprises a pharmaceuticallyacceptable adjuvant, diluent, carrier, preservative, or combinationthereof. In some embodiments, the adjuvant is selected from the groupconsisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, bacillusCalmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone,isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins(KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete),mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols,polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), andcombinations thereof. In some embodiments, the method further comprisesadministering a chemotherapeutic agent, a targeted therapy, radiationtherapy, a checkpoint inhibitor, another immunotherapy, or combinationthereof.

Disclosed herein in some embodiments are human subject-specificpharmaceutical compositions for treatment of a disease or disorder in aspecific human subject, comprising (a) a polypeptide of 10-50 aminoacids in length and comprising a T cell epitope that binds at leastthree HLA class I molecules of the subject and/or at least three HLAclass II molecules of the subject; and (b) a pharmaceutically-acceptableadjuvant. In some embodiments, the composition comprises at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11, or at least 12 differentpolypeptides, each of the different polypeptides being 10-50 amino acidsin length comprising a T cell epitope that binds at least three HLAclass I molecules of the subject and/or at least three HLA class IImolecules of the subject, wherein the amino acid sequence of the T cellepitope of each of the different polypeptides are different from eachother. In some embodiments, the composition comprises 2-40 differentpolypeptides. In some embodiments, the T cell epitope that binds atleast three HLA class I molecules of the subject comprises 7 to 11 aminoacids, and/or the T cell epitope that binds at least three HLA class IImolecules comprises 13 to 17 amino acids. In some embodiments, thecomposition comprises at least two different polypeptides, wherein theepitopes of the at least two different polypeptides are from a singleantigen. In some embodiments, the composition comprises at least twodifferent polypeptides, wherein the epitopes of the at least twodifferent polypeptides are from two or more different antigens. In someembodiments, the antigen is an antigen expressed by a cancer cell, aneoantigen expressed by a cancer cell, a cancer-associated antigen, atumor-associated antigen, or an antigen expressed by a target pathogenicorganism, an antigen expressed by a virus, an antigen expressed by abacterium, an antigen expressed by a fungus, an antigen associated withan autoimmune disorder, or is an allergen.

In some embodiments, the cancer cell is from the subject. In someembodiments, the antigen is selected from the antigens listed in Tables2 to 7. In some embodiments, the composition comprises at least twodifferent polypeptides, wherein two of the polypeptides are arranged endto end or overlapping in a joined polypeptide. In some embodiments, theadjuvant is selected from the group consisting of Montanide ISA-51,QS-21, GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG),corynbacterium parvum, levamisole, azimezone, isoprinisone,dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freundsadjuvant (complete), Freunds adjuvant (incomplete), mineral gels,aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions,oil emulsions, dinitrophenol, diphtheria toxin (DT), and combinationsthereof. In some embodiments, the composition comprises at least twodifferent polypeptides, wherein two of the at least two polypeptides arearranged end to end or overlapping in a joined polypeptide. In someembodiments, the composition comprises two or more different joinedpolypeptides, wherein the two or more different joined polypeptidescomprise different epitopes from each other. In some embodiments, thejoined polypeptides have been screened to eliminate substantially allneoepitopes that span a junction between the two polypeptides and that(i) corresponds to a fragment of a human polypeptide expressed inhealthy cells of the subject; (ii) is a T cell epitope capable ofbinding to at least two HLA class I molecules of the subject; or (iii)meets both requirements (i) and (ii). In some embodiments, the at leasttwo polypeptides do not comprise any amino acid sequences that (i)correspond to a fragment of a human polypeptide expressed in healthycells; or (ii) correspond to a fragment of a human polypeptide expressedin healthy cells and is a T cell epitope capable of binding to at leasttwo HLA class I molecules of the subject.

Disclosed herein in certain embodiments are kits comprising: a firsthuman subject-specific pharmaceutical composition comprising (i) a firstpolypeptide of 10-50 amino acids in length and comprising a T cellepitope that binds at least three HLA class I molecules of the subjectand/or at least three HLA class II molecules of the subject; and (ii) apharmaceutically-acceptable adjuvant; and a second humansubject-specific pharmaceutical composition comprising (i) a secondpolypeptide of 10-50 amino acids in length and comprising a T cellepitope that binds at least three HLA class I molecules of the subjectand/or at least three HLA class II molecules of the subject; and (ii) apharmaceutically-acceptable adjuvant, wherein the first and secondpolypeptides comprise different T cell epitopes. In some embodiments,the first composition and/or the second composition comprise one or moreadditional polypeptides, wherein each additional polypeptide being of10-50 amino acids in length comprising an amino acid sequence that is aT cell epitope that binds at least three HLA class I molecules of thesubject and/or at least three HLA class II molecules of the subject,wherein the amino acid sequences comprise different T cell epitopes.

The disclosure will now be described in more detail, by way of exampleand not limitation, and by reference to the accompanying drawings. Manyequivalent modifications and variations will be apparent, to thoseskilled in the art when given this disclosure. Accordingly, theexemplary embodiments of the disclosure set forth are considered to beillustrative and not limiting. Various changes to the describedembodiments may be made without departing from the scope of thedisclosure. All documents cited herein, whether supra or infra, areexpressly incorporated by reference in their entirety.

The present disclosure includes the combination of the aspects andpreferred features described except where such a combination is clearlyimpermissible or is stated to be expressly avoided. As used in thisspecification and the appended claims, the singular forms “a”, “an”, and“the” include plural referents unless the content clearly dictatesotherwise. Thus, for example, reference to “a peptide” includes two ormore such peptides.

Section headings are used herein for convenience only and are not to beconstrued as limiting in any way.

DESCRIPTION OF THE FIGURES

FIG. 1—ROC curve of HLA restricted PEPI biomarkers.

FIG. 2—ROC curve of ≥1 PEPI3+ Test for the determination of thediagnostic accuracy.

FIGS. 3A-B—Distribution of HLA class I PEPI3+ compared to CD8+ T cellresponses measured by a state of art assay among peptide pools used inthe CD8+ T cell response assays.

FIG. 3A: HLA class I restricted PEPI3+s. The 90% Overall Percent ofAgreement (OPA) among the T cell responses and PEPI3+ peptidesdemonstrate the utility of the disclosed peptides for prediction ofvaccine induced T cell response set of individuals. FIG. 3B: Class I HLArestricted epitopes (PEPI1+). The OPA between predicted epitopes andCD8+ T cell responses was 28% (not statistically significant). Darkestgrey: True positive (TP), both peptide and T cell responses weredetected; Light grey: False negative (FN), only T cell responses weredetected; Lightest grey: False positive (FP), only peptide weredetected; Dark grey: True negative (TN): neither peptides nor T cellresponses were detected.

FIGS. 4A-B—Distribution of HLA class II PEPIs compared to CD4+ T cellresponses measured by a state of art assay among peptide pools used inthe assays. FIG. 4A: HLA class II restricted PEPI4+s. 67% OPA betweenPEPI4+ and CD4+ T-cell responses (p=0.002). FIG. 4B: The class II HLArestricted epitopes. OPA between class II HLA restricted epitopes andCD4+ T cell responses was 66% (not statistically significant). Darkestgrey: True positive (TP), both peptide and T cell responses weredetected; Light grey: False negative (FN), only T cell responses weredetected; Lightest grey: False positive (FP), only peptide weredetected; Dark grey: True negative (TN): neither peptides nor T cellresponses were detected.

FIGS. 5A-D—Multiple HLA binding peptides that define the HPV-16 LPVvaccine specific T cell response set of 18 VIN-3 and 5 cervical cancerpatients. HLA class I restricted PEPI3 counts (FIGS. 5A and 5B) and HLAclass II restricted PEPI3 counts (FIGS. 5C and 5D) derived from LPVantigens of each patient. Light grey: immune responders measured aftervaccination in the clinical trial; Dark grey: Immune non-respondersmeasured after vaccination in the clinical trial. Results show that ≥3HLA class I binding peptides predict the CD8+ T cell reactivity and ≥4HLA class II binding peptides predict the CD4+ T cell reactivity.

FIG. 6—The multiple HLA class I binding peptides that define the HPVvaccine specific T cell response set of 2 patients. Panel A: Four HPVantigens in the HPV vaccine. Boxes represent the length of the aminoacid sequences from the N terminus to the C terminus. Panel B: Processto identify the multiple HLA binding peptides of two patients: HLAsequences of the patients labelled as 4-digit HLA genotype right fromthe patient's ID. The location of the 1^(st) amino acid of the 54 and 91epitopes that can bind to the patient 12-11 and patient 14-5 HLAs(PEPI1+) respectively are depicted with lines. PEPI2 represents thepeptides selected from PEPI1+s that can bind to multiple HLAs of apatient (PEPI2+). PEPI3 represent peptides that can bind to ≥3 HLAs of apatient (PEPI3+). PEPI4 represent peptides that can bind to ≥4 HLAs of apatient (PEPI4+). PEPI5 represent peptides that can bind to ≥5 HLAs of apatient (PEPI5+). PEPI6 represent peptides that can bind to 6 HLAs of apatient (PEPI6). Panel C: The DNA vaccine specific PEPI3+ set of twopatients characterizes their vaccine specific T cell responses.

FIG. 7—Correlation between the ≥1 PEPI3+ Score and CTL response rates ofpeptide targets determined in clinical trials. FIG. 7 discloses SEQ IDNOS 1-4, 6, 5 and 7-13, respectively, in order of appearance.

FIG. 8—Correlation between the ≥1 PEPI3+ Score and the clinical ImmuneResponse Rate (IRR) of immunotherapy vaccines. Dashed lines: 95%confidence band.

FIG. 9—Correlation between the ≥2 PEPI3+ Score and Disease Control Rate(DCR) of immunotherapy vaccines. Dashed lines: 95% confidence band.

FIGS. 10A-D—The IPI-responder HLA Test. Overall Survival (OS) ofmelanoma patients treated with Ipilimumab. Data of 4 independentclinical trials: HLA responders (black line) and HLA non responders(gray line). Statistical analysis: Cox Proportional Hazards SurvivalRegression. FIG. 10A: Trial 1: 18 HLA responders and 30 HLA nonresponders; FIG. 10B: Trial 2: 24 HLA responders and 20 HLA nonresponders; FIG. 10C: Trial 3: 6 HLA responders and 11 HLA nonresponders; FIG. 10D: Trial 4: 13 HLA responders and 38 HLA nonresponders

FIGS. 11A-B—Multiple HLA binding peptides in mutational neoantigens.FIG. 11A: Correlation of mutational load, neoantigen load (neoantigensare neoepitopes according to van Allen) and FIG. 11B: Correlation ofPEPI3+ load and clinical benefit (min-Q1-median-Q3-max).

FIG. 12—HLA map of the Rindopepimut on the HLA alleles of the subjectsin the Model Population. FIG. 12 discloses SEQ ID NO: 87.

FIGS. 13A-B—Probability of vaccine antigen expression in the XYZpatient's tumor cells. There is over 95% probability that 5 out of the12 target antigens in the vaccine regimen is expressed in the patient'stumor. Consequently, the 12 peptide vaccines together can induce immuneresponses against at least 5 ovarian cancer antigens with 95%probability (AGP95). It has 84% probability that each peptide willinduce immune responses in the XYZ patient. AGP50 is the mean (expectedvalue)=7.9 (it is a measure of the effectiveness of the vaccine inattacking the tumor of XYZ patient).

FIG. 14—MRI findings of patient XYZ treated with personalised (PIT)vaccine. This late stage, heavily pretreated ovarian cancer patient hadan unexpected objective response after the PIT vaccine treatment. TheseMRI findings suggest that PIT vaccine in combination with chemotherapysignificantly reduced her tumor burden. The patient now continues thePIT vaccine treatment.

FIGS. 15A-B—Probability of vaccine antigen expression in the ABCpatient's tumor cells. There is over 95% probability that 4 out of the13 target antigens in the vaccine is expressed in the patient's tumor.Consequently, the 12 peptide vaccines together can induce immuneresponses against at least 4 breast cancer antigens with 95% probability(AGP95). It has 84% probability that each peptide will induce immuneresponses in the ABC patient. AGP50 is the mean (expected value) of thediscrete probability distribution=6.45 (it is a measure of theeffectiveness of the vaccine in attacking the tumor of ABC patient).

FIG. 16—Schematic showing exemplary positions of amino acids inoverlapping HLA class I- and HLA class-II binding epitopes in a 30-merpeptide.

DESCRIPTION OF THE SEQUENCES

SEQ ID NOs: 1 to 13 set forth the additional peptide sequences describedin Table 17.

SEQ ID NOs: 14-26 set forth personalised vaccine peptides designed forpatient XYZ described in Table 26.

SEQ ID NOs: 27-38 set forth personalised vaccine peptides designed forpatient ABC described in Table 29.

SEQ ID NOs: 39-86 set forth further 9 mer T cell epitopes described inTable 33.

DETAILED DESCRIPTION HLA Genotypes

HLAs are encoded by the most polymorphic genes of the human genome. Eachperson has a maternal and a paternal allele for the three HLA class Imolecules (HLA-A*, HLA-B*, HLA-C*) and four HLA class II molecules(HLA-DP*, HLA-DQ*, HLA-DRB1*, HLA-DRB3*/4*/5*). Practically, each personexpresses a different combination of 6 HLA class I and 8 HLA class IImolecules that present different epitopes from the same protein antigen.The function of HLA molecules is to regulate T cell responses. Howeverup to date it was unknown how the HLAs of a person regulate T cellactivation.

The nomenclature used to designate the amino acid sequence of the HLAmolecule is as follows: gene name*allele:protein number, which, forinstance, can look like: HLA-A*02:25. In this example, “02” refers tothe allele. In most instances, alleles are defined by serotypes—meaningthat the proteins of a given allele will not react with each other inserological assays. Protein numbers (“25” in the example above) areassigned consecutively as the protein is discovered. A new proteinnumber is assigned for any protein with a different amino acid sequence(e.g. even a one amino acid change in sequence is considered a differentprotein number). Further information on the nucleic acid sequence of agiven locus may be appended to the HLA nomenclature, but suchinformation is not required for the methods described herein.

The HLA class I genotype or HLA class II genotype of an individual mayrefer to the actual amino acid sequence of each class I or class II HLAof an individual, or may refer to the nomenclature, as described above,that designates, minimally, the allele and protein number of each HLAgene. In some embodiments, the HLA genotype of an individual is obtainedor determined by assaying a biological sample from the individual. Thebiological sample typically contains subject DNA. The biological samplemay be, for example, a blood, serum, plasma, saliva, urine, expiration,cell or tissue sample. In some embodiments the biological sample is asaliva sample. In some embodiments the biological sample is a buccalswab sample. An HLA genotype may be obtained or determined using anysuitable method. For example, the sequence may be determined viasequencing the HLA gene loci using methods and protocols known in theart. In some embodiments, the HLA genotype is determined using sequencespecific primer (SSP) technologies. In some embodiments, the HLAgenotype is determined using sequence specific oligonucleotide (SSO)technologies. In some embodiments, the HLA genotype is determined usingsequence based typing (SBT) technologies. In some embodiments, the HLAgenotype is determined using next generation sequencing. Alternatively,the HLA set of an individual may be stored in a database and accessedusing methods known in the art.

HLA-Epitope Binding

A given HLA of a subject will only present to T cells a limited numberof different peptides produced by the processing of protein antigens inan APC. As used herein, “display” or “present”, when used in relation toHLA, references the binding between a peptide (epitope) and an HLA. Inthis regard, to “display” or “present” a peptide is synonymous with“binding” a peptide.

As used herein, the term “epitope” or “T cell epitope” refers to asequence of contiguous amino acids contained within a protein antigenthat possess a binding affinity for (is capable of binding to) one ormore HLAs. An epitope is HLA- and antigen-specific (HLA-epitope pairs,predicted with known methods), but not subject specific. An epitope, a Tcell epitope, a polypeptide, a fragment of a polypeptide or acomposition comprising a polypeptide or a fragment thereof is“immunogenic” for a specific human subject if it is capable of inducinga T cell response (a cytotoxic T cell response or a helper T cellresponse) in that subject. In some cases the helper T cell response is aTh1-type helper T cell response. In some cases an epitope, a T cellepitope, a polypeptide, a fragment of a polypeptide or a compositioncomprising a polypeptide or a fragment thereof is “immunogenic” for aspecific human subject if it is more likely to induce a T cell responseor immune response in the subject than a different T cell epitope (or insome cases two different T cell epitopes each) capable of binding tojust one HLA molecule of the subject.

The terms “T cell response” and “immune response” are used hereininterchangeably, and refer to the activation of T cells and/or theinduction of one or more effector functions following recognition of oneor more HLA-epitope binding pairs. In some cases an “immune response”includes an antibody response, because HLA class II molecules stimulatehelper responses that are involved in inducing both long lasting CTLresponses and antibody responses. Effector functions includecytotoxicity, cytokine production and proliferation. According to thepresent disclosure, an epitope, a T cell epitope, or a fragment of apolypeptide is immunogenic for a specific subject if it is capable ofbinding to at least two, or in some cases at least three, class I or atleast two, or in some cases at least three or at least four class IIHLAs of the subject.

For the purposes of this disclosure we have coined the term “personalepitope”, or “PEPI” to distinguish subject specific epitopes from HLAspecific epitopes. A “PEPI” is a fragment of a polypeptide consisting ofa sequence of contiguous amino acids of the polypeptide that is a T cellepitope capable of binding to one or more HLA class I molecules of aspecific human subject. In other cases a “PEPI” is a fragment of apolypeptide consisting of a sequence of contiguous amino acids of thepolypeptide that is a T cell epitope capable of binding to one or moreHLA class II molecules of a specific human subject. In other words a“PEPI” is a T cell epitope that is recognised by the HLA set of aspecific individual. In contrast to an “epitope”, PEPIs are specific toan individual because different individuals have different HLA moleculeswhich each bind to different T cell epitopes.

“PEPI1” as used herein refers to a peptide, or a fragment of apolypeptide, that can bind to one HLA class I molecule (or, in specificcontexts, HLA class II molecule) of an individual. “PEPI1+” refers to apeptide, or a fragment of a polypeptide, that can bind to one or moreHLA class I molecule of an individual.

“PEPI2” refers to a peptide, or a fragment of a polypeptide, that canbind to two HLA class I (or II) molecules of an individual. “PEPI2+”refers to a peptide, or a fragment of a polypeptide, that can bind totwo or more HLA class I (or II) molecules of an individual, i.e. afragment identified according to a method disclosed herein.

“PEPI3” refers to a peptide, or a fragment of a polypeptide, that canbind to three HLA class I (or II) molecules of an individual. “PEPI3+”refers to a peptide, or a fragment of a polypeptide, that can bind tothree or more HLA class I (or II) molecules of an individual.

“PEPI4” refers to a peptide, or a fragment of a polypeptide, that canbind to four HLA class I (or II) molecules of an individual. “PEPI4+”refers to a peptide, or a fragment of a polypeptide, that can bind tofour or more HLA class I (or II) molecules of an individual.

“PEPI5” refers to a peptide, or a fragment of a polypeptide, that canbind to five HLA class I (or II) molecules of an individual. “PEPI5+”refers to a peptide, or a fragment of a polypeptide, that can bind tofive or more HLA class I (or II) molecules of an individual.

“PEPI6” refers to a peptide, or a fragment of a polypeptide, that canbind to all six HLA class I (or six HLA class II) molecules of anindividual.

Generally speaking, epitopes presented by HLA class I molecules areabout nine amino acids long and epitopes presented by HLA class IImolecules are about fifteen amino acids long. For the purposes of thisdisclosure, however, an epitope may be more or less than nine (for HLAClass I) or more or less than fifteen (for HLA Class II) amino acidslong, as long as the epitope is capable of binding HLA. For example, anepitope that is capable of binding to class I HLA may be between 7, or 8or 9 and 9 or 10 or 11 amino acids long. An epitope that is capable ofbinding to a class II HLA may be between 13, or 14 or 15 and 15 or 16 or17 amino acids long.

Therefore the disclosure herein includes, for example, a method ofpredicting whether a polypeptide is immunogenic for a specific humansubject or identifying a fragment of a polypeptide as immunogenic for aspecific human subject, the method comprising the steps of

-   -   (i) determining whether the polypeptide comprises:        -   a. a sequence of 7 to 11 consecutive amino acids that is            capable of binding to at least two HLA class I of the            subject; or        -   b. a sequence of 13 to 17 consecutive amino acids that is            capable of binding to at least two HLA class II of the            subject; and    -   (ii) predicting that the polypeptide is immunogenic for the        subject if the polypeptide comprises at least one sequence that        meets the requirements of step (i); or predicting that the        polypeptide is not immunogenic for the subject if the        polypeptide does not comprise at least one sequence that meets        the requirements of step (i); or identifying said consecutive        sequence of amino acids as the sequence of a fragment of the        polypeptide that is immunogenic for the subject.

Using techniques known in the art, it is possible to determine theepitopes that will bind to a known HLA. Any suitable method may be used,provided that the same method is used to determine multiple HLA-epitopebinding pairs that are directly compared. For example, biochemicalanalysis may be used. It is also possible to use lists of epitopes knownto be bound by a given HLA. It is also possible to use predictive ormodelling software to determine which epitopes may be bound by a givenHLA. Examples are provided in Table 1. In some cases a T cell epitope iscapable of binding to a given HLA if it has an IC50 or predicted IC50 ofless than 5000 nM, less than 2000 nM, less than 1000 nM, or less than500 nM.

TABLE 1 Example software for determining epitope-HLA binding EPITOPEPREDICTION TOOLS WEB ADDRESS BIMAS, NIHbimas.cit.nih.gov/molbio/hla_bind/ PPAPROC, Tubingen Univ. MHCPred,Edward Jenner Inst. of Vaccine Res. EpiJen, Edward Jenner Inst. ofVaccine ddg-pharmfac.net/epijen/EpiJen/EpiJen.htm Res. NetMHC, Centerfor Biological cbs.dtu.dk/services/NetMHC/ Sequence Analysis SVMHC,Tubingen Univ. abi.inf.uni-tuebingen.de/Services/SVMHC/ SYFPEITHI,Biomedical Informatics,syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm Heidelberg ETKEPITOOLKIT, Tubingen Univ. etk.informatik.uni-tuebingen.de/epipred/PREDEP, Hebrew Univ. Jerusalemmargalit.huji.ac.il/Teppred/mhc-bind/index.html RANKPEP, MIFBioinformatics bio.dfci.harvard.edu/RANKPEP/ IEDB, Immune EpitopeDatabase tools.immuneepitope.org/main/html/tcell_tools.html EPITOPEDATABASES WEB ADDRESS MHCBN, Institute of Microbialimtech.res.in/raghava/mhcbn/ Technology, Chandigarh, INDIA SYFPEITHI,Biomedical Informatics, syfpeithi.de/ Heidelberg AntiJen, Edward JennerInst. of Vaccine ddg-pharmfac.net/antijen/AntiJen/antijenhomepage.htmRes. EPIMHC database of MHC ligands, MIF immunax.dfci.harvard.edu/epimhaBioinformatics IEDB, Immune Epitope Database iedb.org/

As provided herein T cell epitope presentation by multiple HLAs of anindividual is generally needed to trigger a T cell response.Accordingly, the methods of the invention comprise determining whether apolypeptide has a sequence that is a T cell epitope capable of bindingto at least two HLA class I molecules or at least two HLA class II(PEPI2+) molecules of a specific human subject.

The best predictor of a cytotoxic T cell response to a given polypeptideis the presence of at least one T cell epitope that is presented bythree or more HLA class I molecules of an individual (≥1 PEPI3+).Accordingly, in some cases the method comprises determining whether apolypeptide has a sequence that is a T cell epitope capable of bindingto at least three HLA class I molecules of a specific human subject. Insome cases the method comprises determining whether a polypeptide has asequence that is a T cell epitope capable of binding to just three HLAclass I of a specific human subject. A helper T cell response may bepredicted by the presence of at least one T cell epitope that ispresented by three or more (≥1 PEPI3+) or 4 or more (≥1 PEPI4+) HLAclass II of an individual. Therefore in some cases, the method comprisesdetermining whether a polypeptide has a sequence that is a T cellepitope capable of binding to at least three HLA class II of a specifichuman subject. In other cases, the method comprises determining whethera polypeptide has a sequence that is a T cell epitope capable of bindingto at least four HLA class II of a specific human subject. In othercases, the method comprises determining whether a polypeptide has asequence that is a T cell epitope capable of binding to at just threeand/or just four HLA class II of a specific human subject.

In some cases, the disclosure may be used to predict whether apolypeptide/fragment will induce both a cytotoxic T cell response and ahelper T cell response in a specific human subject. Thepolypeptide/fragment comprises both an amino acid sequence that is a Tcell epitope capable of binding to multiple HLA class I molecules of thesubject and an amino acid sequence that is a T cell epitope capable ofbinding to multiple HLA class II molecules of the subject. The HLA classI-binding and HLA class II-binding epitopes may fully or partiallyoverlap. In some cases such fragments of a polypeptide may be identifiedby selecting an amino acid sequence that is a T cell epitope capable ofbinding to at multiple (e.g. at least two or at least three) HLA class Imolecules of the subject, and then screening one or more longerfragments of the polypeptide that are extended at the N- and/orC-terminus for binding to one or more HLA class II molecules of thesubject.

Some subjects may have two HLA alleles that encode the same HLA molecule(for example, two copies for HLA-A*02:25 in case of homozygosity). TheHLA molecules encoded by these alleles bind all of the same T cellepitopes. For the purposes of this disclosure “binding to at least twoHLA molecules of the subject” as used herein includes binding to the HLAmolecules encoded by two identical HLA alleles in a single subject. Inother words, “binding to at least two HLA molecules of the subject” andthe like could otherwise be expressed as “binding to the HLA moleculesencoded by at least two HLA alleles of the subject”.

Polypeptide Antigens

Described herein are methods of predicting whether a polypeptide isimmunogenic for a specific human subject and of identifying a fragmentof a polypeptide as immunogenic for a specific human subject. As usedherein, the term “polypeptide” refers to a full-length protein, aportion of a protein, or a peptide characterized as a string of aminoacids. As used herein, the term “peptide” refers to a short polypeptidecomprising between 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10,or 11, or 12, or 13, or 14, or 15 and 10, or 11, or 12, or 13, or 14, or15, or 20, or 25, or 30, or 35, or 40, or 45, or 50 amino acids.

The terms “fragment” or “fragment of a polypeptide” as used herein referto a string of amino acids or an amino acid sequence typically ofreduced length relative to the or a reference polypeptide andcomprising, over the common portion, an amino acid sequence identical tothe reference polypeptide. Such a fragment according to the disclosuremay be, where appropriate, included in a larger polypeptide of which itis a constituent. In some cases the fragment may comprise the fulllength of the polypeptide, for example where the whole polypeptide, suchas a 9 amino acid peptide, is a single T cell epitope.

In some cases the polypeptide is, or the polypeptide consists of all orpart of an antigen that is, expressed by a pathogenic organism (forexample, a bacteria or a parasite), a virus, or a cancer cell, that isassociated with an autoimmune disorder or response or adisease-associated cell, or that is an allergen, or an ingredient of amedicine or pharmaceutical composition such as a vaccine orimmunotherapy composition. In some cases the method of the disclosurecomprises an initial step of identifying or selecting a suitablepolypeptide, for example a polypeptide as further described below.

The polypeptide or antigen may be expressed in the cells or specificallyin diseased cells of the subject (e.g. a tumor-associated antigen, apolypeptide expressed by a virus, intracellular bacteria or parasite, orthe in vivo product of a vaccine or immunotherapy composition) oracquired from the environment (e.g. a food, an allergen or a drug). Thepolypeptide or antigen may be present in a sample taken from thespecific human subject. Both polypeptide antigens and HLAs can beexactly defined by amino acid or nucleotide sequences and sequencedusing methods known in the art.

The polypeptide or antigen may be a cancer- or tumor-associated antigen(TAA). TAAs are proteins expressed in cancer or tumor cells. The canceror tumour cell may be present in a sample obtained from the subject.Examples of TAAs include new antigens (neoantigens) expressed duringtumorigenesis, products of oncogenes and tumor suppressor genes,overexpressed or aberrantly expressed cellular proteins (e.g. HER2,MUC1), antigens produced by oncogenic viruses (e.g. EBV, HPV, HCV, HBV,HTLV), cancer testis antigens (CTA)(e.g. MAGE family, NY-ESO) andcell-type-specific differentiation antigens (e.g. MART-1). TAA sequencesmay be found experimentally, or in published scientific papers, orthrough publicly available databases, such as the database of the LudwigInstitute for Cancer Research (cta.lncc.br/), Cancer Immunity database(cancerimmunity.org/peptide/) and the TANTIGEN Tumor T cell antigendatabase (cvc.dfci.harvard.edu/tadb/).

In some cases the polypeptide or antigen is not expressed or isminimally expressed in normal healthy cells or tissues, but is expressed(in those cells or tissues) in a high proportion of (with a highfrequency in) subjects having a particular disease or condition, such asa type of cancer or a cancer derived from a particular cell type ortissue, for example breast cancer, ovarian cancer or melanoma. A furtherexample is colorectal cancer. Other non-limiting cancer examples includenon-melanoma skin, lung, prostate, kidney, bladder, stomach, liver,cervix uteri, oesophagus, non-Hodgkin lymphoma, leukemia, pancreas,corpus uteri, lip, oral cavity, thyroid, brain, nervous system,gallbladder, larynx, pharynx, myeloma, nasopharynx, Hodgkin lymphoma,testis and Kaposi sarcoma. Alternatively, the polypeptide may beexpressed at low levels in normal healthy cells, but at high levels(overexpressed) in diseased (e.g. cancer) cells or in subjects havingthe disease or condition. In some cases the polypeptide is expressed in,or expressed at a high level relative to normal healthy cells orsubjects in, at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of suchindividuals, or of a subject-matched human subpopulation. For examplethe subpopulation may be matched to the subject by ethnicity,geographical location, gender, age, disease, disease type or stage,genotype, or expression of one or more biomarkers.

In some cases the expression frequencies can be determined frompublished figures and scientific publications. In some cases the methodof the disclosure comprises a step of identifying or selecting such apolypeptide.

In some cases the polypeptide is associated with or highly (over-)expressed in cancer cells, or in solid tumors. Exemplary cancers includecarcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, orblastomas. The cancer may or may not be a hormone related or dependentcancer (e.g., an estrogen or androgen related cancer). The tumor may bemalignant or benign. The cancer may or may not be metastatic.

In some cases the polypeptide is a cancer testis antigens (CTA). CTA arenot typically expressed beyond embryonic development in healthy cells.In healthy adults, CTA expression is limited to male germ cells that donot express HLAs and cannot present antigens to T cells. Therefore, CTAsare considered expressional neoantigens when expressed in cancer cells.CTA expression is (i) specific for tumor cells, (ii) more frequent inmetastases than in primary tumors and (iii) conserved among metastasesof the same patient (Gajewski ed. Targeted Therapeutics in Melanoma.Springer New York. 2012).

The polypeptide may be a mutational neoantigen, which is expressed by acell, for example a cancer cell, of the individual, but altered from theanalogous protein in a normal or healthy cell. In some cases the methodsof the disclosure comprise the step of identifying a polypeptide that isa mutational neoantigen, or that is a mutational neoantigen in thespecific human subject, or of identifying a neoepitope. For example theneoantigen may be present in a sample obtained from the subject.Mutational neoantigens or neoepitopes can be used to targetdisease-associated cells, such as cancer cells, that express theneoantigen or a neoantigen comprising the neoepitope. Mutations in apolypeptide expressed by a cell, for example a cell in a sample takenfrom a subject, can be detected by, for example, sequencing, but themajority do not induce an immune response against theneoantigen-expressing cells. Currently, the identification of mutationalneoantigens that do induce an immune response is based on prediction ofmutational HLA restricted epitopes and further in vitro testing of theimmunogenicity of predicted epitopes in individual's blood specimen.This process is inaccurate, long and expensive.

As provided herein the identification of mutational epitopes(neoepitopes) that bind to multiple HLA molecules reproducibly definethe immunogenicity of mutational neoantigens. Therefore, in some casesin accordance with the disclosure, the polypeptide is a mutationalneoantigen, and the immunogenic fragment of the polypeptide comprises aneoantigen specific mutation (or consists of a neoepitope).

The polypeptide may be a viral protein that is expressedintracellularly. Examples include HPV16 E6, E7; HIV Tat, Rev, Gag, Pol,Env; HTLV-Tax, Rex, Gag, Env, Human herpes virus proteins, Dengue virusproteins. The polypeptide may be a parasite protein that is expressedintracellularly, for example malaria proteins.

The polypeptide may be an active ingredient of a pharmaceuticalcomposition, such as a vaccine or immunotherapy composition, optionallya candidate active ingredient for a new pharmaceutical composition. Theterm “active ingredient” as used herein refers to a polypeptide that isintended to induce an immune response and may include a polypeptideproduct of a vaccine or immunotherapy composition that is produced invivo after administration to a subject. For a DNA or RNA immunotherapycomposition, the polypeptide may be produced in vivo by the cells of asubject to whom the composition is administered. For a cell-basedcomposition, the polypeptide may be processed and/or presented by cellsof the composition, for example autologous dendritic cells or antigenpresenting cells pulsed with the polypeptide or comprising an expressionconstruct encoding the polypeptide. The pharmaceutical composition maycomprise a polynucleotide or cell encoding one or more active ingredientpolypeptides.

In other cases the polypeptide may be a target polypeptide antigen of apharmaceutical, vaccine or immunotherapy composition. A polypeptide is atarget polypeptide antigen if the composition is intended or designed toinduce an immune response (e.g. a cytotoxic T cell response) thattargets or is directed at the polypeptide. A target polypeptide antigenis typically a polypeptide that is expressed by a pathogenic organism, avirus or a diseased cell such as a cancer cell. A target polypeptideantigens may be a TAA or a CTA.

Presently, >200 clinical trials are investigating cancer vaccines withtumor antigens.

The polypeptide may be an allergen that enters the body of an individualthrough, for example, the skin, lung or oral routes.

Non-limiting examples of suitable polypeptides include those listed inone or more of Tables 2 to 7.

Genetic sequences may be obtained from the sequencing of biologicalmaterials. Sequencing can be done by any suitable method that determinesDNA and/or RNA and/or amino acid sequences. The disclosure utilizes boththe HLA genotypes and amino acid sequences. However, methods to identifyHLA genotype from genetic sequences of an individual and methods ofobtaining amino acid sequences derived from DNA or RNA sequence data arenot the subject of the disclosure.

TABLE 2 LIST OF NAMED TUMOUR ANTIGENS WITH CORRESPONDING ACCESSIONNUMBERS. 5T4 Q13641.1 A1BG P04217.1 A33 Q99795.1 A4GALT Q9NPC4.1 AACTP01011.1 AAG Q9M6E9.1 ABI1 Q8IZP0.1 ABI2 Q9NYB9.1 ABL1 P00519.1 ABL-BCRQ8WUG5.1 ABLIM3 O94929.1 ABLL P42684.1 ABTB1 Q969K4.1 ACACA Q13085.1ACBD4 Q8NC06.1 ACO1 P21399.1 ACRBP Q8NEB7.1* ACTL6A O96019.1 ACTL8Q9H568.1* ACTN4 O43707.1 ACVR1 Q04771.1 ACVR1B P36896.1 ACVR2B Q13705.1ACVRL1 P37023.1 ACS2B Q68CK6.1 ACSL5 Q9ULC5.1 ADAM-15 Q13444.1 ADAM17P78536.1 ADAM2 Q99965.1* ADAM29 Q9UKF5.1* ADAM7 Q9H2U9.1 ADAP1 O75689.1ADFP Q99541.1 ADGRA3 Q8IWK6.1 ADGRF1 Q5T601.1 ADGRF2 Q8IZF7.1 ADGRL2O95490.1 ADHFE1 Q8IWW8.1 AEN Q8WTP8.1 AFF1 P51825.1 AFF4 Q9UHB7.1 AFPP02771.1 AGAP2 Q99490.1 AGO1 Q9UL18.1 AGO3 Q9H9G7.1 AGO4 Q9HCK5.1 AGR2O95994.1 AIFM2 Q9BRQ8.1 AIM2 O14862.1 AKAP-13 Q12802.1 AKAP-3 O75969.1*AKAP-4 Q5JQC9.1* AKIP1 Q9NQ31.1 AKT1 P31749.1 AKT2 P31751.1 AKT3Q9Y243.1 ALDH1A1 P00352.1 ALK Q9UM73.1 ALKBH1 Q13686.1 ALPK1 Q96QP1.1AMIGO2 Q86SJ2.1 ANG2 O15123.1 ANKRD45 Q5TZF3.1* ANO1 Q5XXA6.1 ANP32AP39687.1 ANXA2 P07355.1 APC P25054.1 APEH P13798.1 APOA2 P02652.1 APODP05090.1 APOL1 O14791.1 AR P10275.1 ARAF P10398.1 ARF4L P49703.1 ARHGEF5Q12774.1 ARID3A Q99856.1 ARID4A P29374.1 ARL6IP5 O75915.1 ARMC3B4DXS3.1* ARMC8 Q8IUR7.1 ARTC1 P52961.1 ARX Q96QS3.1* ATAD2 Q6PL18.1ATIC P31939.1 AURKC Q9UQB9.1 AXIN1 O15169.1 AXL P30530.1 BAAT Q14032.1BAFF Q9Y275.1 BAGE-1 Q13072.1* BAGE-2 Q86Y30.1* BAGE-3 Q86Y29.1* BAGE-4Q86Y28.1 BAGE-5 Q86Y27.1* BAI1 O14514.1 BAL P19835.1 BALF2 P03227.1BALF4 P03188.1 BALF5 P03198.1 BARF1 P03228.1 BBRF1 P03213.1 BCANQ96GW7.1 BCAP31 P51572.1 BCL-2 P10415.1 BCL2L1 Q07817.1 BCL6 P41182.1BCL9 O00512.1 BCR P11274.1 BCRF1 P03180.1 BDLF3 P03224.1 BGLF4 P13288.1BHLF1 P03181.1 BHRF1 P03182.1 BILF1 P03208.1 BILF2 P03218.1 BIN1O00499.1 BING-4 O15213.1 BIRC7 Q96CA5.1 BLLF1 P03200.1 BLLF2 P03199.1BMI1 P35226.1 BMLF1 Q04360.1 BMPR1B O00238.1 BMRF1 P03191.1 BNLF2aP0C739.1 BNLF2b Q8AZJ3.1 BNRF1 P03179.1 BRAF1 P15056.1 BRD4 O60885.1BRDT Q58F21.1* BRI3BP Q8WY22.1 BRINP1 O60477.1 BRLF1 P03209.1 BTBD2Q9BX70.1 BUB1B O60566.1 BVRF2 P03234.1 BXLF1 P03177.1 BZLF1 P03206.1C15orf60 Q7Z4M0.1* CA 12-5 Q8WXI7.1 CA 19-9 Q969X2.1 CA195 Q5TG92.1 CA9Q16790.1 CABYR O75952.1* CADM4 Q8NFZ8.1 CAGE1 Q8CT20.1* CALCA P01258.1CALR3 Q96L12.1 CAN P35658.1 CASC3 O15234.1 CASC5 Q8NG31.1* CASP5P51878.1 CASP8 Q14790.1 CBFA2T2 O43439.1 CBFA2T3 O75081.1 CBL P22681.1CBLB Q13191.1 CC3 Q9BUP3.1 CCDC110 Q8TBZ0.1* CCDC33 Q8N5R6.1* CCDC36Q8IYA8.1* CCDC6 Q16204.1 CCDC62 Q6P9F0.1* CCDC68 Q9H2F9.1 CCDC83Q8IWF9.1* CCL13 Q99616.1 CCL2 P13500.1 CCL7 P80098.1 CCNA1 P78396.1*CCNA2 P20248.1 CCNB1 P14635.1 CCND1 P24385.1 CCNE2 O96020.1 CCNIQ14094.1 CCNL1 Q9UK58.1 CCR2 P41597.1 CD105 P17813.1 CD123 P26951.1 CD13P15144.1 CD133 O43490.1 CD137 Q07011.1 CD138 P18827.1 CD157 Q10588.1CD16A P08637.1 CD178 P48023.1 CD19 P15391.1 CD194 P51679.1 CD2 P06729.1CD20 P11836.1 CD21 P20023.1 CD22 P20273.1 CD229 Q9HBG7.1 CD23 P06734.1CD27 P26842.1 CD28 P10747.1 CD30 P28908.1 CD317 Q10589.1 CD33 P20138.1CD350 Q9ULW2.1 CD36 P16671.1 CD37 P11049.1 CD4 P01730.1 CD40 P25942.1CD40L P29965.1 CD45 P08575.1 CD47 Q08722.1 CD51 P06756.1 CD52 P31358.1CD55 P08174.1 CD61 P05106.1 CD70 P32970.1 CD74 P08922.1 CD75 P15907.1CD79B P40259.1 CD80 P33681.1 CD86 P42081.1 CD8a P01732.1 CD8b P10966.1CD95 P25445.1 CD98 P08195.1 CDC123 O75794.1 CDC2 P06493.1 CDC27 P30260.1CDC73 Q6P1J9.1 CDCA1 Q9BZD4.1* CDCP1 Q9H5V8.1 CDH3 P22223.1 CDK2AP1O14519.1 CDK4 P11802.1 CDK7 P50613.1 CDKN1A P38936.1 CDKN2A P42771.1 CEAP06731.1 CEACAM1 Q86UE4.1 CENPK Q9BS16.1 CEP162 Q5TB80.1 CEP290O15078.1* CEP55 Q53EZ4.1* CFL1 P23528.1 CH3L2 Q15782.1 CHEK1 O14757.1CK2 P19784.1 CLCA2 Q9UQC9.1 CLOCK O15516.1 CLPP Q16740.1 CMC4 P56277.1CML66 Q96RS6.1 CO-029 P19075.1 COTL1 Q14019.1 COX2 P35354.1 COX6B2Q6YFQ2.1* CPSF1 Q10570.1 CPXCR1 Q8N123.1* CREBL2 O60519.1 CREG1 O75629.1Cripto P13385.1 CRISP2 P16562.1* *CRK P46108.1 CRKL P46109.1 CRLF2Q9HC73.1 CSAGE Q6PB30.1 CT45 Q5HYN5.1* CT45A2 Q5DJT8.1* CT45A3 Q8NHU0.1*CT45A4 Q8N7B7.1* CT45A5 Q6NSH3.1* CT45A6 P0DMU7.1* CT46 Q86X24.1* CT47Q5JQC4.1* CT47B1 P0C2P7.1* CTAGE2 Q96RT6.1* cTAGE5 O15320.1* CTCFLQ8NI51.1* CTDSP2 O14595.1 CTGF P29279.1 CTLA4 P16410.1 CTNNA2 P26232.1*CTNNB1 P35222.1 CTNND1 O60716.1 CTSH P09668.1 CTSP1 A0RZH4.1* CTTNQ14247.1 CXCR4 P61073.1 CXorf48 Q8WUE5.1* CXorf61 Q5H943.1* Cyclin-EP24864.1 CYP1B1 Q16678.1 CypB P23284.1 CYR61 O00622.1 CS1 P28290.1 CSAG1Q6PB30.1* CSDE1 O75534.1 CSF1 P09603.1 CSF1R P07333.1 CSF3R Q99062.1 CSKP41240.1 CSK23 Q8NEV1.1 DAPK3 O43293.1 DAZ1 Q9NQZ3.1 DBPC Q9Y2T7.1DCAF12 Q5T6F0.1* DCT P40126.1 DCUN1D1 Q96GG9.1 DCUN1D3 Q8IWE4.1 DDR1Q08345.1 DDX3X O00571.1 DDX6 P26196.1 DEDD O75618.1 DEK P35659.1 DENRO43583.1 DEPDC1 Q5TB30.1 DFNA5 O60443.1 DGAT2 Q96PD7.1 DHFR P00374.1DKK1 O94907.1 DKK3 Q9UBP4.1 DKKL1 Q9UK85.1* DLEU1 O43261.1 DMBT1Q9UGM3.1 DMRT1 Q9Y5R6.1* DNAJB8 Q8NHS0.1* DNAJC8 O75937.1 DNMT3AQ9Y6K1.1 DPPA2 Q7Z7J5.1* DR4 O00220.1 DRS O14763.1 DRG1 Q9Y295.1* DSCR8Q96T75.1 E2F3 O00716.1 E2F6 O75461.1 E2F8 A0AVK6.1 EBNA1 P03211.1 EBNA2P12978.1 EBNA3 P12977.1 EBNA4 P03203.1 EBNA6 P03204.1 EBNA-LP Q8AZK7.1E-cadherin P12830.1 ECT2 Q9H8V3.1 ECTL2 Q008S8.1 EDAG Q9BXL5.1* EEF2P13639.1 EFNA1 P20827.1 EF5 O43281.1 EFTUD2 Q15029.1 EGFL7 Q9UHF1.1 EGFRp00533.1 EI24 O14681.1 EIF4EBP1 Q13541.1 ELF3 P78545.1 ELF4 Q99607.1ELOVL4 Q9GZR5.1* EMP1 P54849.1 ENAH Q8N8S7.1 Endosialin Q9HCU0.1 ENO1P06733.1 ENO2 P09104.1 ENO3 P13929.1 ENTPD5 O75356.1 EpCAM P16422.1EPHA2 P29317.1 EPHA3 P29320.1 EPHB2 P29323.1 EPHB4 P54760.1 EPHB6O15197.1 EPS8 Q12929.1 ERBB3 P21860.1 ERBB4 Q15303.1 EREG O14944.1 ERGP11308.1 ERVK-18 O42043.1 ERVK-19 O71037.1 ESR1 P03372.1 ETAA1 Q9NY74.1ETS1 P14921.1 ETS2 P15036.1 ETV1 P50549.1 ETV5 P41161.1 ETV6 P41212.1EVI5 O60447.1 EWSR1 Q01844.1 EYA2 O00167.1 EZH2 Q15910.1 FABP7 O15540.1FAM133A Q8N9E0.1* FAM13A O94988.1 FAM46D Q8NEK8.1* FAM58BP P0C7Q3.1FANCG O15287.1 FATE1 Q969F0.1* FBXO39 Q8N4B4.1* FBXW11 Q9UKB1.1 FCHSD2O94868.1 FER P16591.1 FES P07332.1 FEV Q99581.1 FGF10 O15520.1 FGF23Q9GZV9.1 FGF3 P11487.1 FGF4 P08620.1 FGF5 P12034.1 FGFR1 P11362.1 FGFR2P21802.1 FGFR3 P22607.1 FGFR4 P22455.1 FGR P09769.1 FLI1 Q01543.1 FLT3P36888.1 FMNL1 O95466.1 FMOD Q06828.1 FMR1NB Q8NOW7.1* FN1 P02751.1 Fn14Q9NP84.1 FNIP2 Q9P278.1 FOLR1 P15328.1 FOS P01100.1 FosB P53539.1 FOSL1P15407.1 FOXM1 Q08050.1 FOXO1 Q12778.1 FOXO3 O43524.1 FRAT1 Q92837.1FRMD3 A2A2Y4.1 FSIP1 Q8NA03.1 FSIP2 Q5CZC0.1 FSTL3 O95633.1 FTHL17Q9BXU8.1* FUNDC2 Q9BWH2.1 FUS P35637.1 FUT1 P19526.1 FUT3 P21217.1 FYNP06241.1 GAB2 Q9UQC2.1 GADD45G O95257.1 GAGE-1 Q13065.1 GAGE12B/C/D/EGAGE12F P0CL80. 1 GAGE12G P0CL81.1 GAGE12H A6NDE8. 1 A1L429.1 GAGE12IP0CL82.1 GAGE12J A6NER3. 1 GAGE-2 Q6NT46.1 GAGE-3 Q13067.1 GAGE-4Q13068.1 GAGE-5 Q13069.1 GAGE-6 Q13070.1 GAGE-7 O76087.1 GAGE-8 Q9UEU5.1GALGT2 Q00973.1 GAS7 O60861.1 GASZ Q8WWH4.1 GATA-3 P23771.1 GBU4-5Q587J7.1 GCDFP-15 P12273.1 GFAP P14136.1 GFI1 Q99684.1 Ghrelin Q9UBU3.1GHSR Q92847.1 GIPC1 O14908.1 GITR Q9Y5U5.1 GKAP1 Q5VSY0.1 GLI1 P08151.1Glypican-3 P51654.1 GML Q99445.1 GNA11 P29992.1 GNAQ P50148.1 GNB2L1P63244.1 GOLGA5 Q8TBA6.1 gp100 P40967.1 gp75 P17643.1 Gp96 P14625.1GPAT2 Q6NUI2.1* GPATCH2 Q9NW75.1* GPC-3 P51654.1 GPNMB Q14956.1 GPR143P51810.1 GPR89A B7ZAQ6.1 GRB2 P62993.1 GRP78 P11021.1 GUCY1A3 Q02108.1H3F3A P84243.1 HAGE Q9NXZ2.1* hANP P01160.1 HBEGF Q99075.1 hCG-betaP01233.1 HDAC1 Q13547.1 HDAC2 Q92769.1 HDAC3 O15379.1 HDAC4 P56524.1HDAC5 Q9UQL6.1 HDAC6 Q9UBN7.1 HDAC7 Q8WUI4.1 HDAC8 Q9BY41.1 HDAC9Q9UKV0.1 HEATR1 Q9H583.1 Hepsin P05981.1 Her2/neu P04626.1 HERC2O95714.1 HERV-K104 P61576.1 HEXB P07686.1 HEXIM1 O94992.1 HGRG8 Q9Y5A9.1HIPK2 Q9H2X6.1 HJURP Q8NCD3.1 HMGB1 P09429.1 HMOX1 P09601.1 HNRPLP14866.1 HOM-TES-85 Q9P127.1* HORMAD1 Q86X24.1* HORMAD2 Q8N7B1.1* HPSEQ9Y251.1 HPV16 E6 P03126.1 HPV16 EV P03129.1 HPV18 E6 P06463.1 HPV18 E7P06788.1 HRAS P01112.1 HSD17B13 Q7Z5P4.1 HSP105 Q92598.1 HSP60 P10809.1HSPA1A P08107.1 HSPB9 Q9BQS6.1* HST-2 P10767.1 HT001 Q2TB18.1 hTERTO14746.1 HUS1 O60921.1 ICAM-1 P05362.1 IDH1 O75874.1 IDO1 P14902.1 IER3P46695.1 IGF1R P08069.1 IGFS11 Q5DX21.1* IL13RA2 Q14627.1* IMP-3Q9NV31.1* ING3 Q9NXR8.1 INPPL1 O15357.1 INTS6 Q9UL03.1 IRF4 Q15306.1IRS4 O14654.1 ITGA5 P08648.1 ITGB8 P26012.1 ITPA Q9BY32.1 ITPR2 Q14571.1JAK2 O60674.1 JAK3 P52333.1 JARID1B Q9UGL1.1* JAZF1 Q86VZ6.1 JNK1P45983.1 JNK2 P45984.1 JNK3 P53779.1 JTB O76095.1 JUN P05412.1 JUPP14923.1 K19 P08727.1 KAAG1 Q9UBP8.1 Kallikrein 14 Q9P0G3.1 Kallikrein 4Q9Y5K2.1 KAT6A Q92794.1 KDM1A O60341.1 KDM5A P29375.1 KIAA0100 Q14667.1*KIAA0336 Q8IWJ2.1 KIAA1199 Q8WUJ3.1 KIAA1641 A6QL64.1 KIF11 P52732.1KIF1B O60333.1 KIF20A O95235.1 KIT P10721.1 KLF4 O43474.1 KLHL41O60662.1 KLK10 O43240.1 KMT2D O14686.1 KOC1 O00425.1 K-ras P01116.1KRIT1 O00522.1 KW-12 P62913.1 KW-2 Q96RS0.1 KW-5 (SEBD4) Q9H0Z9.1 KW-7O75475.1 L1CAM P32004.1 L53 Q96EL3.1 L6 Q9BTT4.1 LAG3 P18627.1 Lage-1O75638.1* LATS1 O95835.1 LATS2 Q9NRM7.1 LCMT2 O60294.1 LCP1 P13796.1LDHC P07864.1* LDLR P01130.1 LEMD1 Q68G75.1* Lengsin Q5TDP6.1 LETMD1Q6P1Q0.1 LGALS3BP Q08380.1 LGALS8 O00214.1 LIN7A O14910.1 LIPI Q6XZB0.1*LIV-1 Q13433.1 LLGL1 Q15334.1 LMO1 P25800.1 LMO2 P25791.1 LMP1 P03230.1LMP2 P13285.1 LOC647107 Q8TAI5.1* LOXL2 Q9Y4K0.1 LRP1 Q07954.1 LRRN2O75325.1 LTF P02788.1 LTK P29376.1 LZTS1 Q9Y250.1 LY6K Q17RY6.1* LYNP07948.1 LYPD6B Q8NI32.1* MAEA Q7L5Y9.1 MAEL Q96JY0.1* MAF O75444.1 MAFFQ9ULX9.1 MAFG O15525.1 MAFK O60675.1 MAGE-A1 P43355.1* MAGE-A10P43363.1* MAGE-A11 P43364.1* MAGE-A12 P43365.1* MAGE-A2 P43356.1*MAGE-A2B Q6P448.1* MAGE-A3 P43357.1* MAGE-A4 P43358.1* MAGE-A5 P43359.1*MAGE-A6 P43360.1* MAGE-A8 P43361.1* MAGE-A9 P43362.1* MAGE-B1 P43366.1*MAGE-B2 O15479.1* MAGE-B3 O15480.1* MAGE-B4 O15481.1* MAGE-B5 Q9BZ81.1*MAGE-B6 Q8N7X4.1* MAGE-C1 O60732.1* MAGE-C2 Q9UBF1.1* MAGE-C3 Q8TD91.1*mammaglobin-A MANF P55145.1 MAP2K2 P36507.1 MAP2K7 O14733.1 Q13296.1MAP3K7 O43318.1 MAP4K5 Q9Y4K4.1 MARTI Q16655.1 MART-2 Q5VTY9.1 MAS1P04201.1 MC1R Q01726.1 MCAK Q99661.1* MCF2 P10911.1 MCF2L O15068.1 MCL1Q07820.1 MCTS1 Q9ULC4.1 MCSP Q6UVK1.1 MDK P21741.1 MDM2 Q00987.1 MDM4O15151.1 ME1 P48163.1 ME491 P08962.1 MECOM Q03112.1 MELK Q14680.1 MEN1O00255.1 MERTK Q12866.1 MET P08581.1 MFGE8 Q08431.1 MFHAS1 Q9Y4C4.1 MFI2P08582.1 MGAT5 Q09328.1 Midkine P21741.1 MIF P14174.1 MKI67 P46013.1MLH1 P40692.1 MLL Q03164.1 MLLT1 Q03111.1 MLLT10 P55197.1 MLLT11Q13015.1 MLLT3 P42568.1 MLLT4 P55196.1 MLLT6 P55198.1 MMP14 P50281.1MMP2 P08253.1 MMP7 P09237.1 MMP9 P14780.1 MOB3B Q86TA1.1 MORC1 Q86VD1.1*MPHOSPH1 Q96Q89.1* MPL P40238.1 MRAS O14807.1 MRP1 P33527.1 MRP3O15438.1 MRPL28 Q13084.1 MRPL30 Q8TCC3.1 MRPS11 P82912.1 MSLN Q13421.1MTA1 Q13330.1 MTA2 O94776.1 MTA3 Q9BTC8.1 MTCP1 P56278.1 MTSS1 O43312.1MUC-1 P15941.1 MUC-2 Q02817.1 MUC-3 Q02505.1 MUC-4 Q99102.1 MUC-5ACP98088.1 MUC-6 Q6W4X9.1 MUM1 Q2TAK8.1 MUM2 Q9Y5R8.1 MYB P10242.1 MYCP01106.1 MYCL P12524.1 MYCLP1 P12525.1 MYCN P04198.1 MYD88 Q99836.1MYEOV Q96EZ4.1 MYO1B O43795.1 NA88-A P0C5K6.1* NAE1 Q13564.1 Napsin-AO96009.1 NAT6 Q93015.1 NBAS A2RRP1.1 NBPF12 Q5TAG4.1 NCOA4 Q13772.1NDC80 O14777.1 NDUFC2 O95298.1 Nectin-4 Q96NY8.1 NEK2 P51955.1 NEMFO60524.1 NENF Q9UMX5.1 NEURL1 O76050.1 NFIB O00712.1 NFKB2 Q00653.1NF-X1 Q12986.1 NFYC Q13952.1 NGAL P80188.1 NGEP Q6IWH7.1 NKG2D-L1Q9BZM6.1 NKG2D-L2 Q9BZM5.1 NKG2D-L3 Q9BZM4.1 NKG2D-L4 Q8TD07.1 NKX3.1Q99801.1 NLGN4X Q8N0W4.1 NLRP4 Q96MN2.1* NNMT P40261.1 NOL4 O94818.1*NOTCH2 Q04721.1 NOTCH3 Q9UM47.1 NOTCH4 Q99466.1 NOV P48745.1 NPM1P06748.1 NR6A1 Q15406.1* N-RAS P01111.1 NRCAM Q92823.1 NRP1 O14786.1NSE1 Q96KN4.1 NSE2 Q96KN1.1 NTRK1 P04629.1 NUAK1 O60285.1 NUGGC Q68CJ6.1NXF2 Q9GZY0.1* NXF2B Q5JRM6.1* NY-BR-1 Q9BXX3.1 NYD-TSPG Q9BWV7.1NY-ESO-1 P78358.1* NY-MEL-1 P57729.1 OCA2 Q04671.1 ODF1 Q14990.1* ODF2Q5BJF6.1* ODF3 Q96PU9.1* ODF4 Q2M2E3.1* OGG1 O15527.1 OGT O15294.1 OIP5O43482.1* OS9 Q13438.1 OTOA Q05BM7.1* OX40 P43489.1 OX40L P23510.1 P53P04637.1 P56-LCK P06239.1 PA2G4 Q9UQ80.1 PAGE1 O75459.1* PAGE2 Q7Z2X2.1*PAGE2B Q5JRK9.1* PAGE3 Q5JUK9.1* PAGE4 O60829.1* PAGE5 Q96GU1.1* PAK2Q13177.1 PANO1 I0J062.1 PAP Q06141.1 PAPOLG Q9BWT3.1 PARK2 O60260.1PARK7 Q99497.1 PARP12 Q9H0J9.1 PASD1 Q8IV76.1* PAX3 P23760.1 PAX5Q02548.1 PBF P00751.1 PBK Q96KB5.1* PBX1 P40424.1 PCDC1 Q15116.1 PCM1Q15154.1 PCNXL2 A6NKB5.1 PDGFB P01127.1 PDGFRA P16234.1 PEPP2 Q9HAU0.1*PGF P49763.1 PGK1 P00558.1 PHLDA3 Q9Y5J5.1 PHLPP1 O60346.1 PIAS1O75925.1 PIAS2 O75928.1 PIK3CA P42336.1 PIK3CD O00329.1 PIK3R2 O00459.1PIM1 P11309.1 PIM2 Q9P1W9.1 PIM3 Q86V86.1 PIR O00625.1 PIWIL1 Q96J94.1*PIWIL2 Q8TC59.1* PIWIL3 Q7Z3Z3.1 PIWIL4 Q7Z3Z4.1 PKN3 Q6P5Z2.1 PLA2G16P53816.1 PLAC1 Q9HBJ0.1* PLAG1 Q6DJT9.1 PLEKHG5 O94827.1 PLK3 Q9H4B4.1PLS3 P13797.1 PLVAP Q9BX97.1 PLXNB1 O43157.1 PLXNB2 O15031.1 PMLP29590.1 PML-RARA Q96QH2.1 POTEA Q6S8J7.1* POTEB Q6S5H4.1* POTECB2RU33.1* POTED Q86YR6.1* POTEE Q6S8J3.1* POTEG Q6S5H5.1* POTEHQ6S545.1* PP2A P63151.1 PPAPDC1B Q8NEB5.1 PPFIA1 Q13136.1 PPIG Q13427.1PPP2R1B P30154.1 PRAME P78395.1* PRDX5 P30044.1 PRKAA1 Q13131.1 PRKCIP41743.1 PRM1 P04553.1* PRM2 P04554.1* PRMT3 O60678.1 PRMT6 Q96LA8.1PDL1 Q9NZQ7.1 PROM1 O43490.1 PRSS54 Q6PEW0.1* PRSS55 Q6UWB4.1* PRTN3P24158.1 PRUNE Q86TP1.1 PRUNE2 Q8WUY3.1 PSA P07288.1 PSCA D3DWI6.1 PSMAQ04609.1 PSMD10 O75832.1 PSGR Q9H255.1 PSP-94 Q1L6U9.1 PTEN P60484.1PTH-rP P12272.1 PTK6 Q13882.1 PTPN20A Q4JDL3.1* PTPRK Q15262.1 PTPRZP23471.1 PTTG-1 O95997.1 PTTG2 Q9NZH5.1 PTTG3 Q9NZH4.1 PXDNL A1KZ92.1RAB11FIP3 O75154.1 RAB8A P61006.1 RAD1 O60671.1 RAD17 O75943.1 RAD51CO43502.1 RAF1 P04049.1 RAGE-1 Q9UQ07.1 RAP1A P62834.1 RARA P10276.1RASSF10 A6NK89.1 RB1 P06400.1 RBL2 Q08999.1 RBM46 Q8TBY0.1* RBP4P02753.1 RCAS1 O00559.1 RCVRN P35243.1 RECQL4 O94761.1 RET P07949.1RGS22 Q8NE09.1* RGS5 O15539.1 RHAMM O75330.1 RhoC P08134.1 RHOXF2Q9BQY4.1 RL31 P62888.1 RNASET2 O00584.1 RNF43 Q68DV7.1 RNF8 O76064.1 RONQ04912.1 ROPN1A Q9HAT0.1* ROR1 Q01973.1 RPA1 O95602.1 RPL10A P62906.1RPL7A P62424.1 RPS2 P15880.1 RPS6KA5 O75582.1 RPSA P08865.1 RQCD1Q92600.1* RRAS2 P62070.1 RSL1D1 O76021.1 RTKN Q9BST9.1 RUNX1 Q01196.1RUNX2 Q13950.1 RYK P34925.1 SAGE1 Q9NXZ1.1* SART2 Q9UL01.1 SART3Q15020.1 SASH1 O94885.1 sCLU P10909.1 SCRN1 Q12765.1 SDCBP O00560.1SDF-1 P48061.1 SDHD O14521.1 SEC31A O94979.1 SEC63 Q9UGP8.1 Semaphorin4D SEMG1 P04279.1* SFN P31947.1 SH2B2 O14492.1 Q92854.1 SH2D1B O14796.1SH3BP1 Q9Y3L3.1 SHB Q15464.1 SHC3 Q92529.1 SIRT2 Q8IXJ6.1 SIVA1 O15304.1SKI P12755.1 SLBP A9UHW6.1 SLC22A10 Q63ZE4.1 SLC25A47 Q6Q0C1.1 SLC35A4Q96G79.1 SLC45A3 Q96JT2.1 SLC4A1AP Q9BWU0.1 SLCO6A1 Q86UG4.1* SLITRK6Q9H5Y7.1 Sm23 P27701.1 SMAD5 Q99717.1 SMAD6 O43541.1 SMO Q99835.1 Smt3BP61956.1 SNRPD1 P62314.1 SOS1 Q07889.1 SOX-2 P48431.1 SOX-6 P35712.1SOX-11 P35716.1 SPA17 Q15506.1* SPACA3 Q8IXA5.1* SPAG1 Q07617.1* SPAG17Q6Q759.1* SPAG4 Q9NPE6.1* SPAG6 O75602.1* SPAG8 Q99932.1* SPAG9O60271.1* SPANXA1 Q9NS26.1* SPANXB Q9NS25.1* SPANXC Q9NY87.1* SPANXDQ9BXN6.1* SPANXE Q8TAD1.1* SPANXN1 Q5VSR9.1* SPANXN2 Q5MJ10.1* SPANXN3Q5MJ09.1* SPANXN4 Q5MJ08.1* SPANXN5 Q5MJ07.1* SPATA19 Q7Z5L4.1* SPEF2Q9C093.1* SPI1 P17947.1 SPINLW1 O95925.1* SPO11 Q9Y5K1.1* SRC P12931.1SSPN Q14714.1 SSX-1 Q16384.1* SSX-2 Q16385.1* SSX-3 Q99909.1* SSX-4O60224.1* SSX-5 O60225.1* SSX-6 Q7RTT6.1* SSX-7 Q7RTT5.1* SSX-9Q7RTT3.1* ST18 O60284.1 STAT1 P42224.1 STEAP1 Q9UHE8.1 STK11 Q15831.1STK25 O00506.1 STK3 Q13188.1 STN Q9H668.1 SUPT7L O94864.1 SurvivinO15392.1 SUV39H1 O43463.1 SYCE1 Q8NOS2.1 SYCP1 Q15431.1 SYCP3 Q8IZU3.1SYT Q15532.1 TA-4 Q96RI8.1 TACC1 O75410.1 TAF1B Q53T94.1 TAF4 O00268.1TAF7L Q5H9L4.1* TAG-1 Q02246.1* TALI P17542.1 TAL2 Q16559.1 TAPBPO15533.1 TATI P00995.1 TAX1BP3 O14907.1 TBC1D3 Q8IZP1.1 TBP-1 P17980.1TCL1A P56279.1 TCL1B O95988.1 TDHP Q9BT92.1 TDRD1 Q9BXT4.1* TDRD4Q9BXT8.1* TDRD6 O60522.1* TEKT5 Q96M29.1* TEX101 Q9BY14.1* TEX14Q8IWB6.1* TEX15 Q9BXT5.1* TEX38 Q6PEX7.1* TF P02787.1 TFDP3 Q5H9I0.1*TFE3 P19532.1 TGFBR1 P36897.1 TGFBR2 P37173.1 THEG Q9P2T0.1* TIE2Q02763.1 TIPRL O75663.1 TLR2 O60603.1 TMEFF1 Q8IYR6.1* TMEFF2 Q9UIK5.1*TMEM108 Q6UXF1.1* TMEM127 O75204.1 TMPRSS12 Q86WS5.1* TNC P24821.1TNFRSF17 Q02223.1 TNFSF15 O95150.1 TNK2 Q07912.1 TOMM34 Q15785.1 TOP2AP11388.1 TOP2B Q02880.1 TOR3A Q9H497.1 TP73 O15350.1 TPA1 8N543.1 TPGS2Q68CL5.1 TPI1 P60174.1 TPL2 P41279.1 TPM4 P67936.1 TPO P40225.1 TPPP2P59282.1* TPR P12270.1 TPTE P56180.1* TRAF5 O00463.1 TRAG-3 Q9Y5P2.1*TRGC2 P03986.1 TRIM24 O15164.1 TRIM37 O94972.1 TRIM68 Q6AZZ1.1 TRPM8Q7Z2W7.1 TSGA10 Q9BZW7.1* TSP50 Q9UI38.1* TSPAN6 O43657.1 TSPY1Q01534.1* TSPY2 A6NKD2.1* TSPY3 Q6B019.1* TSPYL1 Q9H0U9.1 TSSK6Q9BXA6.1* TTC23 Q5W5X9.1 TTK P33981.1* TULP2 O00295.1* TUSC2 O75896.1TWEAK O43508.1 TXNIP Q9H3M7.1 TYMS P04818.1 TYR P14679.1 U2 snRNP BP08579.1 U2AF1 Q01081.1 UBD O15205.1 UBE2A P49459.1 UBE2C O00762.1UBE2V1 Q13404.1 UBE4B O95155.1 UBR5 O95071.1 UBXD5 Q5T124.1 UFL1O94874.1 URI1 O94763.1 URLC10 Q17RY6.1 UROC1 Q96N76.1 USP2 O75604.1 USP4Q13107.1 VAV1 P15498.1 VCX3A Q9NNX9.1 VEGFR1 P17948.1 VEGFR2 P35968.1VHL P40337.1 VIM P08670.1 VWA5A O00534.1 WHSC2 Q9H3P2.1 WISP1 O95388.1WNK2 Q9Y351.1 WNT10B O00744.1 WNT3 P56703.1 WNT-5a P41221.1 WT1 P19544.1WWP1 Q9HOM0.1 XAGE-1 Q9HD64.1* XAGE-2 Q96GT9.1* XAGE-3 Q8WTP9.1* XAGE-4Q8WWM0.1 XAGE-5 Q8WWM1.1* XBP1 P17861.1 XPO1 O14980.1 XRCC3 O43542.1YB-1 P67809.1 YEATS4 O95619.1 YES1 P07947.1 YKL-40 P36222.1 ZBTB7AO95365.1 ZBTB7C A1YPR0.1 ZEB1 P37275.1 ZFYVE19 Q96K21.1 ZNF165 P49910.1*ZNF185 O15231.1 ZNF217 O75362.1 ZNF320 A2RRD8.1 ZNF395 Q9H8N7.1 ZNF645Q8N7E2.1* ZUBR1 Q5T457.1 ZW10 O43264.1 ZWINT O95229.1 CTAs = bold and *

TABLE 3 LIST OF ACCESSION NUMBERS FOR VIRAL ANTIGENS FROM IEDB Q76R62.1P03182.1 P09258.1 P09310.1 P03227.1 P89466.1 P04601.1 P13285.1 P09991.1P03468.1 A2T3Q0.1 P0C6X7.1 P89448.1 P12978.1 P09257.1 P50641.1 P14075.120178567.1 Q01023.1 P03188.1 P04585.1 P0C767.1 P12977.1 P89467.1Q9W850.1 Q00683.1 P04591.1 P03211.1 9628706.1 P03460.1 P08666.1 P03485.1Q04360.1 Q913Y7.1 P89449.1 Q81871.1 P03452.1 P17763.1 P89430.1 P03410.1P04012.1 P27958.1 Q6WB99.1 P25212.1 Q9PZT1.1 P68593.1 P03203.1 P29996.19629374.1 P59633.1 O42053.1 P0C6L3.1 P59635.1 Q9YZN9.1 Q6WB95.1 P10233.1P89475.1 Q6WB98.1 Q6SW67.1 Q7TFA0.1 P0CK17.1 P59594.1 1980491.1 P14079.1P15423.1 1891762.1 P09259.1 P09269.1 Q77Q38.1 Q786F2.1 Q6SW99.1 P24771.1F5HB98.1 9629370.1 P68336.1 P03300.1 1980486.1 Q69027.1 P28284.1P13290.1 9626585.1 P06923.1 P14076.1 P03346.1 O42062.1 P07566.1 P03204.1Q69091.1 P09255.1 P03206.1 O36634.1 P10205.1 F5HCM1.1 P0CK16.1 Q6WB97.1Q85601.1 P89468.1 Q69467.1 P03218.1 Q786F3.1 P59637.1 1891763.1 Q6WB94.1P03231.1 Q91K92.1 Q6WBA1.1 P03466.1 P14335.1 P26670.1 Q9PZT0.1 1985356.1Q2HR63.1 P59634.1 Q6SW59.1 P03277.1 P59595.1 Q69028.1 P03383.1 P03261.1P03200.1 P04578.1 P06484.1 F5HC97.1 S5TC82.1 P18095.1 Q96895.1 P18094.19629372.1 P50791.1 P03230.1 P13845.1 9629712.1 P03209.1 P03129.1Q76R61.1 P03228.1 P0C206.1 Q9WMB5.1 P03226.1 Q9QR69.1 O36633.1 O42049.1P03496.1 P03428.1 P03431.1 P0C0U1.1 P03433.1 P03508.1 1980456.1 P0C739.1P69726.1 P69723.1 1980490.1 532129755.1 P03120.1 P04020.1 P06922.1P03114.1 P03314.1 P06790.1 P06788.1 P06927.1 P03101.1 P03107.1 P06794.1530787712.1 P04013.1 Q80872.1 P04014.1 P03126.1 P36811.1 P06463.1P26554.1 P04016.1 P14078.1 P03191.1 1980471.1 P06821.1 P0C797.1 F5HF49.1P0C045.1 P04296.1 P04485.1 P10230.1 P10221.1 P06487.1 P10215.1 P04293.1P10211.1 P10209.1 P10225.1 P10224.1 P10238.1 P10185.1 P08392.1 P10231.1P06492.1 P04290.1 P08393.1 P08543.1 P10210.1 P08617.1 F5HB53.1 P04019.1P04015.1 P89442.1 P89452.1 P89462.1 P59632.1 O36635.1 P07210.1 Q83884.1Q8JUX5.1 P03089.1 Q66479.1 P03185.1 P0CAP6.1 P04618.1 56160929.11980519.1 P08669.1 P14348.1 P03212.1 P03179.1 45617.1 1511872.1302317869.1 P69899.1 P09247.1 Q05127.1 P18272.1 Q9YMG2.1 Q05128.1302371215.1 302371218.1 Q5XX08.1 302371214.1 P14336.1 138948.1 P08292.11803956.1 P35253.1 1891726.1 P09308.1 P03189.1 667489389.1 P09272.134365530.1 Q05320.1 P59596.1 P32886.1 55097.1 P03316.1 P03276.1 Q81870.1Q81862.1 64320.1 1933190.1

TABLE 4 LIST OF ACCESSION NUMBERS FOR BACTERIAL ANTIGENS FROM IEDBB8ZUD1.1 P09621.1 P9WPE5.1 Q2GI62.1 P0A5B8.1 O50443.1 Q5NEZ3.1 P9WQF5.1P9WK95.1 O05311.1 P9WQD7.1 P9WKG3.1 P9WHE5.1 P0CD83.1 P9WHB9.1 P9WH91.1P9WHE3.1 P9WNK7.1 A0A0F3MKF3.1 A1JIP3.1 B2RKS6.1 P0A1D3.1 P0A6F5.1P0C0Z7.1 P0C923.1 P61439.1 Q9Z708.1 P0A521.1 P9WPE7.1 Q79FJ2.1 B8ZR84.1I6Y3P5.1 Q2FYP2.1 P9WG41.1 P96890.1 O06625.1 I6X654.1 Q8YIE1.1 P9WQ81.1I6XWA1.1 P11311.1 O53900.1 P9WIR7.1 P9WQB1.1 B8ZUC6.1 O06802.1 P9WMK1.1P9WG37.1 Q2FWC4.1 Q2GGE3.1 O33347.1 P9WJ09.1 P9WJ11.1 P9WF23.1 O69703.1I6X4K0.1 B2RM93.1 P71888.1 P9WFW3.1 P9WPV1.1 P9WPU7.1 P9WPV3.1 P9WPU5.1O50391.1 P9WID7.1 P9WPC3.1 P96901.1 O84848.1 Q2FUX4.1 A0A0M1YNY3.1P49944.1 P9WPQ9.1 Q45010.1 Q2FZK7.1 P9WMN3.1 P9WPQ1.1 Q45013.1 O53666.1Q5NEH1.1 P9WHR5.1 P9WIE5.1 Q5NEQ3.1 P9WNF3.1 F2QBN0.1 B8ZTB7.1 P0C922.1P9WMJ9.1 Q5NGW2.1 P01556.1 Q8DMZ4.1 P33768.1 Q2FUY2.1 Q5NG56.1 X8CE55.1Q5NGE4.1 P94973.1 O06827.1 P96872.1 I6X9Y7.1 I6XFZ8.1 O50442.1 O53697.1O53978.1 P95137.1 P95144.1 O53519.1 Q79FZ8.1 P9WJF5.1 P71629.1 P9WJS3.1P9WPB7.1 Q7D9T1.1 P9WHS1.1 O06393.1 P9WP69.1 P9WPN5.1 P9WNX3.1 O53380.1I6YAU3.1 P0A4V2.1 P9WQP3.1 P0C2T2.1 P9WQP1.1 P9WQN9.1 O53311.1 P9WIS7.1O06159.1 H2GU79.1 Q2G2Q0.1 P9WNV1.1 P9WNV5.1 Q8YE98.1 Q59191.1 P9WGY7.1P9WGY9.1 Q2G2W1.1 P9WGH1.1 P9WNG9.1 P9WNG7.1 O84591.1 Q9Z7A6.1 P9WGR1.1P96404.1 I6YGS0.1 Q6MX18.1 P9WNK5.1 O53692.1 P9WNK3.1 P9WNK1.1 P9WNJ9.1P9WNJ7.1 P9WNJ5.1 P9WNJ3.1 P9WNJ1.1 P9WNI9.1 P96903.1 P9WNB1.1 P9WJE1.1P9WJD9.1 P9WJD7.1 P9WJD3.1 P9WJC5.1 P9WJC3.1 P9WJC1.1 P9WNQ3.1 P9WJE5.1P9WJC7.1 O84646.1 I6YDV4.1 P11439.1 Q5NFJ1.1 P9WNE5.1 P14738.1 P11089.1H7C7G3.1 L7N6B9.1 I6XFI7.1 O05578.1 P96218.1 P9WN39.1 P9WN59.1 Q8YBI3.1P9WN83.1 P9WJA9.1 P9WMY9.1 Q5NH51.1 O53673.1 P9WIP9.1 P0CE15.1 P72041.1Q5NEM8.1 Q5NI16.1 P9WJA3.1 P0A4Q1.1 P9WIP1.1 P9WIN9.1 P9WNF5.1 O50846.1Q59947.1 H7C7N8.1 Q5NEC6.1 O84606.1 P9WQJ9.1 P9WQJ7.1 P9WQ71.1 O53611.1P9WKL1.1 P9WKJ7.1 D5V9Y8.1 P0CC04.1 P23700.1 P9WJN5.1 Q5NHJ0.1 Q5NEY9.1P15917.1 Q2G155.1 O34094.1 Q8F8E1.1 O69661.1 H6MMU4.1 P9WK61.1 P9WK55.1Q8YGS9.1 O50811.1 P9WQ59.1 P9WIN7.1 P9WIR1.1 O50430.1 D5VCH6.1 Q5NHI7.1P9WFU9.1 I6XFY8.1 B2RH54.1 Q46409.1 P30690.1 A0A0J5IWN3.1 A0PSI5.1A4TAC4.1 B1MB69.1 B2HSY2.1 B8ZSN3.1 E4WHS0.1 P9WK17.1 V5XE39.1 I6X7G8.1I6Y461.1 I6YGB1.1 I6YC99.1 Q79FY7.1 I6X5Z8.1 I6Y479.1 I6YA32.1 O05461.1Q2G1E2.1 P9WK19.1 I6YAW3.1 Q5NGG4.1 O51624.1 P9WJW5.1 Q50584.1 B2RHG1.1Q5NFL7.1 P9WQN7.1 P9WHH3.1 O84639.1 Q5NF24.1 P9WJH1.1 P9WJH5.1 O53203.1P55969.1 O50418.1 Q5NGE0.1 H7C7K8.1 O54584.1 G1UB30.1 Q5NH85.1 G1UB25.1P0A3N8.1 E1X6Y5.1 Q5NEP7.1 Q8YHH0.1 P38006.1 P43838.1 P43839.1 P0CL67.1P0CL66.1 Q0SLZ0.1 Q07337.1 G5IXI6.1 O07721.1 O53254.1 P75330.1 I6Y936.1L7N649.1 L7N656.1 L7N693.1 Q79FK4.1 Q79FR3.1 Q79FR5.1 Q79G04.1 Q79FS8.1Q6MWX1.1 Q79FV6.1 Q79FS5.1 Q79FQ7.1 Q79FP3.1 Q79FP2.1 Q79FK9.1 Q79FE6.1I6XEF1.1 Q79FD4.1 Q6MX26.1 Q6MX50.1 L7N680.1 O53695.1 I6X8R2.1 O53246.1I6Y0L1.1 Q2G282.1 P14283.1 P04977.1 P9WMX7.1 P9WFR1.1 P9WN09.1 O86345.1P9WGU1.1 P9WGT9.1 P9WGT7.1 P9WPF7.1 P9WIB3.1 P9WMM9.1 P9WHM5.1 P9WQE9.1Q8DQ08.1 Q8DQ07.1 I6Y231.1 P9WHV9.1 O05877.1 O07236.1 O86370.1 O06404.1O06410.1 B8ZRL2.1 O06807.1 O33269.1 Q79FA9.1 Q79FK6.1 Q8VKN2.1 L7N675.1Q79FK5.1 L0T7Y7.1 Q79F19.1 Q79FE1.1 Q6MWX9.1 O84616.1 O84647.1 P9WQ27.1O84288.1 I6X9S5.1 P9WJW3.1 P9WPS9.1 P95149.1 O53632.1 I6Y293.1 L0T243.1P9WP43.1 P9WKC9.1 P96402.1 P71810.1 O06417.1 P96365.1 L0T5B2.1 P96264.1P9WJK5.1 P9WJQ9.1 O84419.1 O84818.1 Q8YG32.1 O06608.1 O07175.1 P9WGA3.1O53323.1 P96354.1 P9WIM9.1 B8ZRT2.1 P9WK93.1 P13423.1 O84583.1 P9WG63.1P9WIM1.1 P9WKJ3.1 P9WNZ7.1 P9WK31.1 Q50701.1 P9WID3.1 Q8YC41.1 P9WPL3.1P9WNI3.1 P9WNI7.1 P9WNI5.1 P9WQ49.1 P9WMG1.1 Q2GGR3.1 P9WK71.1 O33192.1P9WND5.1 P9WFL9.1 P9WMB7.1 P9WJ79.1 P9WND7.1 Q63RA7.1 Q63ID0.1 I6YET7.1Q9S010.1 P9WGC9.1 Q50700.1 Q5NFR6.1 P9WGK3.1 P9WHI1.1 P9WHV3.1 Q5NIA7.1P9WG27.1 P9WF73.1 P9WGA1.1 P9WIB9.1 P9WGL3.1 O51381.1 P9WI83.1 P9WI79.1P9WFT7.1 Q8YGS6.1 P05788.1 P17835.1 P9WIK9.1 Q5NHP7.1 P9WJU5.1 P9WGE7.1Q2G2B2.1 P04958.1 P9WG67.1 P9WKE1.1 O07226.1 P9WJ13.1 P9WHF3.1 P9WF43.1Q7D7L0.1 P9WMF9.1 P9WGN1.1 P9WKJ9.1 P60230.1 P9WKH7.1 O53699.1 P9WHT7.1P9WJS5.1 Q5NII0.1 Q8YDZ3.1 Q9RPX7.1 P9WN67.1 O05576.1 Q5NHL4.1 P9WN15.1P9WMD5.1 P9WMF5.1 P9WG85.1 P9WJW7.1 P9WIH1.1 P9WIG1.1 P9WIG3.1 P9WIF5.1P9WIF1.1 P9WIE7.1 P9WHW9.1 P9WI41.1 P9WI39.1 P9WI37.1 P9WI25.1 Q11031.1P9WI47.1 P9WI23.1 P9WI19.1 P9WI11.1 P9WI45.1 P9WI07.1 P9WI05.1 Q79FH3.1P9WI43.1 P9WHZ7.1 P9WHZ5.1 P9WHZ3.1 P9WHY9.1 P9WHY7.1 P9WHY5.1 Q6MX07.1P9WHY3.1 Q6MWY2.1 Q50703.1 P9WHX3.1 P96221.1 Q7D589.1 P9WMA3.1 P9WKW1.1P9WKS9.1 P9WM29.1 P9WGC1.1 P9WLZ5.1 P9WLZ3.1 P9WLX1.1 P9WLV9.1 P9WLS7.1P9WLQ1.1 P9WLJ1.1 P9WLH9.1 P9WLF3.1 P9WL97.1 P9WL87.1 P9WL85.1 P9WL83.1P9WL67.1 P9WL63.1 P9WL51.1 P9WL47.1 P9WNH3.1 P9WGL7.1 P9WQM5.1 P9WPD9.1A0A098A1N7.1 A0A098A2B0.1 A2RGM0.1 A5LVF6.1 A5MKZ9.1 B8ZQI8.1 B8ZQM3.1B8ZQT5.1 B8ZR82.1 B8ZRH1.1 B8ZS71.1 B8ZS85.1 B8ZS86.1 B8ZSJ5.1 B8ZSL3.1B8ZSL7.1 B8ZSM6.1 B8ZT30.1 B8ZTD0.1 B8ZTS2.1 B8ZTV5.1 B8ZU53.1 B8ZUA4.1B8ZUE5.1 B8ZUF0.1 B8ZUT6.1 B8ZUX6.1 C0R9U8.1 C6DPT8.1 C6DQ35.1 E1XJN6.1G8W6L3.1 G8W6L7.1 G8W6U7.1 H6MNY3.1 H6MQD5.1 H8HRN0.1 H8HW90.1 H8L8K3.1I6TQ53.1 I6TX52.1 P0C5B9.1 Q1BYS7.1 R4MDK6.1 S5F815.1 W6GWM1.1 P9WFC9.1P9WFJ9.1 P14916.1 P69996.1 P9WFC5.1 Q8VKQ6.1 P9WHS3.1 A5MKI6.1

TABLE 5 LIST OF ACCESSION NUMBERS FOR FUNGAL ANTIGENS FROM IEDB andUNIPROT Q5ANA3.1 Q5A3P6.1 Q59VM7.1 Q5A1A9.1 Q5APF0.1 Q8J0P4.1 Q4WHG0.1Q4WQ87.1 Q59X67.1 Q59Z17.1 Q59ZI3.1 Q5AA33.1 B8N4Q9.1 Q4WAW6.1 Q4WAJ6.1Q4X1V0.1 A0A1D8PQ86.1 Q59ZB1.1 Q873N2.1 Q59L72.1 B8NIF0.1 P46075.1Q4WCL1.1 Q4WRP2.1 Q59L12.1 Q59LC9.1 P48989.1 Q5AFC2.1 B8N406.1 Q4WGL5.1Q9HEQ8.1 Q4WVI6.1 P46593.1 P82611.1 Q5ADV5.1 Q59SG9.1 P41750.1 O00092.1Q4WEN1.1 Q4WCV3.1 P0DJ06.1 O94038.1 Q59WD3.1 Q59RQ0.1 B8NM71.1 Q4WLW8.1Q4WI37.1 Q4WNI1.1 P29717.1 P46589.1 Q59W04.1 Q59RK9.1 B8MYS6.1 Q8X176.1Q4WZS1.1 Q4WQH4.1 Q9UW14.1 Q5AF56.1 Q59VN0.1 P31353.1 B8N8Q9.1 Q96UX3.1Q4WDA4.1 Q4WDE1.1 Q92207.1 P83773.1 Q59WB9.1 Q5ACM4.1 B8N8R3.1 Q4WPF5.1Q4WLS7.1 Q4WJT7.1 Q5A8T7.1 Q59YU1.1 Q59P53.1 Q5ACI8.1 B8N417.1 Q92450.1Q4WWM6.1 Q4WLG1.1 Q5A8T4.1 Q59YV2.1 Q5A432.1 Q5AB93.1 B8N8R0.1 Q4WAW9.1Q4WP81.1 Q4WQR6.1 P43076.1 Q5ABE5.1 Q5AK64.1 Q5ALL8.1 B8NM74.1 A4GYZ0.1Q6MYT0.1 Q4WZS2.1 Q5AP53.1 Q59LF2.1 A0A1D8PNZ7.1 Q5A4X8.1 B8N106.1Q4WAW3.1 Q4WTL0.1 Q4WXP0.1 Q5AL52.1 Q8NJN3.1 Q59Q30.1 Q5AD34.1 B8NHY4.1Q70J59.1 Q4WXV2.1 Q4WU59.1 P43079.1 Q5ALN1.1 A0A1D8PN12.1 Q59V02.1B8NJG8.1 Q4X1A4.1 Q4X0Z3.1 Q4WUG4.1 Q5AD07.1 Q59S72.1 Q5AK24.1 Q5AHC0.1B8NM66.1 E9R876.1 Q4WN25.1 Q4WIK9.1 Q5A0E5.1 Q59K86.1 Q5AFT2.1 Q59Y11.1B8MYL0.1 M4VQY9.1 Q4WN21.1 Q4WYP0.1 Q5AKU6.1 Q5AGD1.1 Q5A0W6.1 Q59QA5.1B8NM62.1 Q4WF53.1 Q4X1N0.1 Q4X0B5.1 Q59RL7.1 P79023.1 P0CB63.1 Q5AMJ5.1B8NGT5.1 Q4WZ64.1 Q4WQV2.1 Q4WYK9.1 G1UB61.1 Q59LP6.1 Q59U11.1 Q5AMF7.1B8NM64.1 Q4WAZ0.1 Q4WZP2.1 Q4WY33.1 Q5ABC6.1 Q5AP87.1 P83775.1 Q5ABW2.1B8NV37.1 Q4WR16.1 Q4WVK2.1 Q4X1F8.1 A0A1D8PQB9.1 P22274.1 Q5APF2.1Q5APJ9.1 B8N151.1 Q4WLB9.1 Q4WUA0.1 Q4WA45.1 P87020.1 Q5AC48.1 Q59VP2.1Q5AM72.1 B8NEJ3.1 Q4WQS0.1 A4DA84.1 Q4WKD7.1 P0CY27.1 Q5AP59.1 Q5AEE1.1Q5ACU3.1 B8N8M2.1 Q4WEP7.1 Q4WJX0.1 Q4WCH5.1 Q59XX2.1 Q59MV1.1 Q5AMR5.1Q5A1V3.1 B8MYV0.1 E9R9Y3.1 Q4WP38.1 Q4WXY3.1 Q59U10.1 Q5AL27.1 Q59SU5.1Q59RF7.1 B8N7I7.1 P41748.1 Q4X1D7.1 Q4WPL7.1 Q59RW5.1 Q5AJD2.1 Q59VP1.1Q5ACN3.1 B8NJG3.1 Q4WYG3.1 Q4W9Z9.1 Q4X136.1 Q59MQ0.1 P0CU38.1 Q5ADQ0.1Q5AHE8.1 B8N8R1.1 P87184.1 Q4WE62.1 Q4WZ44.1 Q5ABU7.1 Q59QC5.1 Q5AK59.1Q5AHA4.1 B8NJH2.1 Q4WBS1.1 Q4WZL3.1 Q4WTC7.1 Q9Y7F0.1 Q5A5N6.1 Q59RH5.1Q5AEG7.1 B8NQ51.1 Q70DX9.1 Q4WB37.1 Q4WMK2.1 Q5AC08.1 Q59Q79.1 Q5ACW8.1Q59V01.1 B8NM63.1 Q4WG16.1 Q4W9Z4.1 Q4WNC9.1 P30575.1 Q5AH38.1 Q5AGM0.1Q5AK97.1 B8NM73.1 Q96X30.1 Q4WDD0.1 Q4WY67.1 Q5AAG6.1 Q5AMN3.1 Q59VN2.1Q5A1B2.1 B8NYX0.1 Q4WV19.1 Q4WKB9.1 Q4WU12.1 O74189.1 Q5A1Z5.1 O94069.1Q5AJK6.1 B8N3P7.1 Q4WAZ6.1 Q4WU07.1 Q4WA61.1 Q59W62.1 Q5A6K2.1 P0CY20.1Q59L96.1 B8NJH1.1 Q4W944.1 Q4WBL6.1 Q4WA58.1 P0CY34.1 Q59L25.1 Q59XQ1.1Q59MD0.1 B8MXJ7.1 Q4WTV7.1 Q4WX13.1 Q4WA60.1 Q5A1D3.1 Q5A922.1 O94048.1Q5AG46.1 B8NJB0.1 Q4WMJ9.1 Q4WV71.1 Q4WX36.1 Q5AJU7.1 Q5AFG1.1 Q5ADX2.1Q59VW6.1 B8NPS7.1 Q4WZ65.1 Q4X0C2.1 Q4WA62.1 Q5A4H5.1 Q5ALR8.1 P46586.1Q5A8I6.1 B8N7Z8.1 A0A067Z9B6.1 Q4WRU4.1 Q4WA59.1 Q59Y31.1 Q5AEI2.1P83776.1 Q9UW24.1 B8NSV5.1 Q66WM4.1 Q4WGS4.1 Q4WXQ7.1 P0CY29.1 Q5AI71.1Q5A895.1 Q59Q38.1 B8MZA3.1 Q6T267.1 Q4WP13.1 Q4WVA0.1 Q5ANJ4.1 Q5ABA6.1Q59PP0.1 Q5ADL0.1 B8NLY9.1 Q4WLW5.1 Q4WHG5.1 Q4WDN4.1 Q59NH8.1 Q5ABX0.1Q5AHH4.1 Q5AH11.1 B8NR69.1 Q4WMJ0.1 Q4WPF7.1 Q4WK03.1 P0CY33.1 Q5A4N0.1Q96UX5.1 Q59W55.1 B8MZ41.1 Q4WQUO.1 Q4WH83.1 Q4WCG2.1 Q00310.1 Q59TN9.1P87206.1 Q5AC37.1 B8N7S7.1 Q4WMJ8.1 Q4WXW1.1 Q4WX99.1 Q5A0W9.1 Q5A5S7.1Q5A029.1 Q5A7Q3.1 B8NR71.1 Q4WWN8.1 Q8NJM2.1 Q4WV10.1 Q5A4M8.1 Q59UG3.1Q5A1E0.1 Q59PV6.1 A0A0D9MRV9.1 Q4WZ63.1 Q4WWD3.1 Q4WIS6.1 Q5AJC0.1P0C075.1 Q59XL0.1 P0CH96.1 P55790.1 Q4WVN4.1 Q4WPU8.1 Q4WP65.1 Q59SU1.1Q59R09.1 Q5A6U1.1 P83782.1 B8NM72.1 Q4WAY8.1 Q4WN99.1 Q4WUK1.1 Q5AG71.1Q9B8D4.1 Q5A8I8.1 Q5A660.1 B8MW78.1 Q4WY07.1 P0C959.1 Q4WKN3.1 Q5AMT2.1Q9B8D3.1 Q59PR9.1 Q59YT1.1 Q9P900.1 Q4WZ66.1 Q4X0S7.1 Q4WG58.1 Q59KY8.1Q9B8D5.1 O74261.1 P53709.1 B8NDE2.1 Q4WQZ5.1 Q4WPW2.1 Q4WXX9.1 Q59LY1.1Q59LR2.1 Q96VB9.1 Q5ACX1.1 B8NJF4.1 O42630.1 Q4X1U0.1 Q4WC37.1 Q59UT4.1Q5AED9.1 Q5AQ47.1 Q5ADP9.1 B8NIV9.1 POC7S9.1 Q4WP57.1 Q4X1Y0.1 Q5ABC5.1Q5A4W8.1 Q5A985.1 Q92210.1 B8NG16.1 Q4WI46.1 Q4WPH9.1 Q4WZL8.1 Q59MV9.1Q5ANH2.1 Q59ZW2.1 Q59MA3.1 B8NX60.1 Q4WQY4.1 Q4WDK5.1 Q4WR80.1 Q59MD2.1Q5A649.1 P83784.1 Q5AFK3.1 B8NM75.1 Q4WAY3.1 Q4WI71.1 Q4WY53.1 Q5A8N2.1Q5AI22.1 Q59P11.1 Q59S63.1 B8MZZ6.1 Q4WT66.1 Q4WYS7.1 Q4WL88.1 P40953.1Q5A950.1 Q5ADN8.1 Q5A0Y2.1 B8NM67.1 Q6MY57.1 Q4WY08.1 Q4WGV9.1 Q5APR8.1Q5ANC9.1 Q5A849.1 Q5ALW7.1 B8NRX2.1 P0C954.1 Q4WND3.1 Q4WC29.1 P10613.1Q59UH7.1 Q5A7R7.1 Q59W52.1 B8NXJ2.1 Q4W946.1 Q4X1D2.1 Q4WKV8.1 Q5A5Q6.1Q5ALX8.1 Q59XB0.1 Q59S42.1 B8NMD3.1 Q4WMJ5.1 Q6MY91.1 Q4WYA5.1 Q5A4F3.1Q5AI37.1 Q59P96.1 Q5A961.1 B8NBI2.1 Q70GH4.1 Q4WRV2.1 Q4WCM6.1 P43094.1Q5ABV4.1 Q59SR6.1 Q59ST6.1 B8NPA4.1 Q4WUL6.1 Q4WRX4.1 Q4WKB2.1 Q9P940.1Q5AKU4.1 Q9P975.1 Q59N74.1 B8N803.1 P61832.1 Q4WP03.1 Q4WNG7.1 Q5AJY5.1Q59VY1.1 O94083.1 Q5A6P6.1 B8NPT0.1 Q4WG11.1 Q4WTA6.1 Q4WRE8.1 P39827.1Q59Z51.1 Q5AIA4.1 Q59XM0.1 B8MXP5.1 Q4WYU4.1 Q4WZJ0.1 Q9P8P4.1 Q59WF4.1Q59LV8.1 Q59YF4.1 Q5A4N5.1 B8NIB8.1 Q4WYR6.1 Q4W9S8.1 Q4WJS4.1 P83774.1Q59X11.1 Q59XW9.1 Q5A6M2.1 B8N9H4.1 Q4WNE1.1 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B8N219.1Q4WX94.1 Q4WLS7.1 Q4X1I8.1 Q5AG77.1 P87163.1 Q5A8A2.1 Q59Q36.1 B8NQK0.1Q4WLD0.1 Q4WWM6.1 Q4WVW4.1 Q9UW13.1 Q5AI86.1 Q9B8D7.1 Q92410.1 Q12732.1Q4WUK5.1 Q4WP81.1 Q4WTH1.1 P0CU34.1 Q5AM80.1 Q9UW25.1 Q5A1M4.1 Q9HEY7.1Q8TGG5.1 Q6MYT0.1 Q4WLI9.1 P40954.1 Q5A6Q7.1 Q59XY9.1 Q5ANC8.1 Q6UEG8.1Q4WTK9.1 Q4WTL0.1 Q4WQJ5.1 Q04802.1 Q5AGV4.1 Q5A210.1 Q5A4K7.1 O427T6.1Q4WVU5.1 Q4WXV2.1 Q4WQJ2.1 P0CY35.1 Q5AJ82.1 Q5AGW8.1 Q5ADL8.1 Q9UW95.1Q4WLM7.1 Q4X0Z3.1 Q4WK56.1 Q5AAU5.1 Q5AIA1.1 Q5ADS3.1 Q59RQ2.1 Q9Y8D9.1Q4W9P4.1 Q4WN25.1 Q4WJS2.1 Q59VQ8.1 Q5A9Z6.1 Q5ACR4.1 Q5APC0.1 A2SZW8.1Q4WIT0.1 Q4WN21.1 Q4WJT9.1 Q59VF4.1 Q5AGC1.1 P0CU36.1 Q5A931.1 Q2U2U3.1Q4WQB9.1 Q4X1N0.1 Q4WUV8.1 Q5A0X8.1 Q59ZV5.1 Q5A2Y7.1 Q59VW7.1 Q00258.1Q4WGK6.1 Q4WQV2.1 Q4WX68.1 O13426.1 Q59VP7.1 Q5A368.1 Q5AKU5.1 Q12437.1Q4WMR0.1 Q4WZP2.1 Q4WHN8.1 Q5A0M4.1 Q5A7P3.1 Q9B8D6.1 Q59MN0.1 E9QYP0.1Q4WYE5.1 Q4WVK2.1 Q4WJU8.1 Q59PF9.1 Q5A6K8.1 Q9B8D0.1 Q59WH7.1 Q4WS76.1Q4WZ01.1 Q4WUA0.1 Q4WBT4.1 Q5AFP3.1 Q5AD13.1 Q5A2K0.1 Q96WL3.1 Q4WMJ7.1Q4W930.1 A4DA84.1 Q4WZV6.1 Q5AEK8.1 Q04782.1 Q5A1Q5.1 Q59ZX6.1 P28296.1Q4WBR0.1 Q4WJX0.1 Q4WUV9.1 Q5AFK0.1 Q5A0J9.1 Q5AEM5.1 Q59MU1.1 E9RAH5.1Q4WHD1.1 Q4WP38.1 Q4WLV2.1 Q5APD4.1 Q59ZZ6.1 Q5AK25.1 Q5A0J0.1 Q4WW81.1Q4WTB3.1 Q4X1D7.1 Q4WFS2.1 Q5ADQ9.1 Q5AH25.1 Q5AK10.1 Q59WK2.1 Q50EL0.1Q4WRV9.1 Q4W9Z9.1 Q4WBM1.1 P83779.1 Q59XM1.1 Q5AI15.1 P43073.1 Q4WY82.1Q4X267.1 Q4WE62.1 Q4WAU7.1 Q5AAH2.1 Q59NN8.1 Q5AEM8.1 P87220.1 Q4WSF6.1Q4WVZ3.1 Q4WZL3.1 Q4WZS3.1 O74254.1 Q5AP65.1 Q5A4J4.1 Q5ABD9.1 E9RCK4.1Q4WR24.1 Q4WB37.1 Q4WPU9.1 Q5AL49.1 Q5AFF7.1 Q59YK4.1 P83781.1 Q4WZA8.1Q4WPM8.1 Q4W9Z4.1 Q4WVZ0.1 P53697.1 Q59VR3.1 Q59WV0.1 Q5ANB1.1 Q4WAW7.1Q4WE86.1 Q4WDD0.1 Q4WCX9.1 Q5ACL7.1 Q5AFH3.1 Q5AHB1.1 Q5A0E2.1 Q92405.1A4DA70.1 Q4WKB9.1 Q4WJ38.1 Q5AEM6.1 P83780.1 Q5APK0.1 Q5AMG5.1 Q4WRY5.1Q4WW45.1 Q4WU07.1 Q4WRC2.1 Q8TG40.1 Q5A4G9.1 Q59PW0.1 Q5A6T8.1 Q7Z7W6.1Q4WVG2.1 Q4WBL6.1 Q4WWW5.1 Q59X38.1 Q59NQ9.1 O74711.1 Q59WG5.1 Q4WZ67.1Q4WQG9.1 Q4WX13.1 Q4WC84.1 Q59VQ3.1 A0A1D8PNP3.1 Q5ADN9.1 Q5AI80.1Q4WZB3.1 Q4WQN1.1 Q4WV71.1 Q4WTW3.1 Q5A7Q2.1 Q5A9Z1.1 Q5ACP5.1 Q5AB49.1Q4WLN1.1 Q4WCF1.1 Q4X0C2.1 Q4WFV6.1 Q5AJV5.1 A0A1D8PK89.1 Q5A1E1.1Q59R32.1 Q4WR82.1 Q4WZC3.1 Q4WRU4.1 Q4WKD9.1 Q5A3Z6.1 Q59WB3.1 Q59L86.1Q5A061.1 O14434.1 Q4WYX7.1 Q4WGS4.1 Q4WP10.1 Q5A201.1 Q59ZC8.1 Q5AD23.1Q59P50.1 Q4WMK0.1 Q4X0A5.1 Q4WP13.1 C5JZM2.1 O93827.1 Q5A1L6.1 Q5A5U6.1Q59WC6.1 Q4WPX2.1 Q4WUD3.1 Q4WHG5.1 P0DJ06.1 Q5AAI8.1 A0A1D8PN14.1Q5ADQ7.1 Q5AI48.1 O43099.1 Q4WS49.1 Q4WPF7.1 P46598.1 Q5A2J7.1 Q5A8X7.1Q59WJ4.1 Q59ZU1.1 Q4WJ81.1 Q4WCX7.1 Q4WH83.1 P87020.1 P22011.1 Q59X39.1Q5AGV7.1 Q5AG56.1 P67875.1 Q4WXX5.1 Q4WXW1.1 P38110.1 Q9HGT6.1 Q5ACW6.1Q59NR8.1 Q59T36.1 Q4WZB4.1 Q4WNB5.1 Q8NJM2.1 C1GK29.1 Q9UW26.1 P0CB54.1Q5A5K7.1 Q9P840.1 E9QUT3.1 O42799.1 Q4WWD3.1 Q59LX5.1 A0A1D8PN88.1Q5A210.1 Q5AHB8.1 Q4WAZ9.1 Q4WHA3.1 Q4WPU8.1 Q59PT0.1 A0A1D8PMB1.1Q59N10.1 Q5AKU3.1 Q4WZ70.1 Q4W9M3.1 Q4WN99.1 Q3MNT0.1 Q5ABR2.1 Q5A1B3.1Q59ZW4.1 E9RBR0.1 Q4WVH5.1 P0C959.1

TABLE 6 LIST OF ACCESSION NUMBERS FOR ALLERGENS FROM IEDB &ALLERGENONLINE P19594.1 P28335.1 P29000.1 M5ECN9.1 P38948.1 P00709.1P79085.1 P49148.1 Q6R4B4.1 P42037.1 Q9HDT3.1 P42058.1 P0C0Y4.1 P27759.1Q2KN25.1 P00304.1 Q2KN24.1 Q2KN27.1 P43174.1 P10414.1 Q8L5L5.1 Q8GZP6.1Q8H2B8.1 Q7Z1K3.1 A1IKL2.1 Q7M1X6.1 P49372.1 P00630.1 P43238.1 Q45W87.1Q6PSU2.1 O82580.1 Q647G9.1 Q9SQH1.1 C7E3T4.1 H6VGI3.1 Q84ZX5.1 A0PJ16.1P67875.1 P40292.1 P28296.1 P79017.1 Q96X30.1 Q4WWX5.1 O60024.1 Q92450.1Q09072.1 Q09097.1 P04403.1 P15494.1 P25816.1 P43187.1 Q39419.1 O65002.1P05814.1 P13916.1 Q9UAM5.1 P54958.1 D0VNY7.1 P54962.1 O18598.1 Q1A7B3.1Q9NG56.1 A0ERA8.1 Q8MUF6.1 A7IZE9.1 O96870.1 P02663.1 P02666.1 P02668.1Q28133.1 P00711.1 P02754.1 P02769.1 P02662.1 O18873.1 P49822.1 P09582.1B5KVH4.1 Q14790.1 E9R5X9.1 Q96385.1 Q7M1E7.1 P02229.1 Q7XCK6.1 P40108.1P42039.1 P42040.1 P42059.1 P0C0Y5.1 P02465.1 Q6IQX2.1 P20023.1 Q08407.1Q8S4P9.1 Q9ATH2.1 Q8W1C2.1 P18632.1 P43212.1 Q9SCG9.1 Q9M4S6.1 Q69CS2.1Q96VP3.1 O04701.1 O04725.1 P94092.1 P04800.1 Q7M1X8.1 Q41183.1 P93124.1P82946.1 O04298.1 Q58A71.1 Q23939.1 Q967Z0.1 Q1M2P5.1 Q94507.1 Q8MVU3.1Q86R84.1 Q00855.1 P49275.1 Q26456.1 P08176.1 Q8N0N0.1 P49278.1 Q2L7C5.1P39675.1 Q9Y197.1 P14004.1 P49273.1 Q7Z163.1 Q9UL01.1 O15315.1 P11388.1P30575.1 Q95182.1 P41091.1 O15371.1 P25780.1 Q2PS07.1 P49327.1 P30438.1Q5VFH6.1 Q7XAV4.1 P04075.1 Q90YL0.1 P01005.1 P01012.1 P19121.1 P02230.1P02224.1 P02227.1 Q9NJQ6.1 O65809.1 P26987.1 P04776.1 P04347.1 P04405.1P08238.1 P12031.1 P15252.1 Q7Y1X1.1 P52407.1 O82803.1 Q39967.1 P02877.1P62805.1 P43216.1 O23972.1 P24337.1 Q7Y1C1.1 P93198.1 Q9SEW4.1 Q2TPW5.1P81294.1 P81295.1 O64943.1 P07498.1 Q84UI1.1 P80384.1 P31025.1 Q004B5.1P14946.1 Q7M1X5.1 P14947.1 P14948.1 Q5TIW3.1 Q40237.1 P14174.1 Q5H786.1P30440.1 P11589.1 P43211.1 P40967.1 Q01726.1 Q16655.1 Q07932.1 Q9ZNZ4.1Q9H009.1 P12036.1 Q15233.1 Q5RZZ3.1 Q8GZB0.1 Q8NFH4.1 P19963.1 Q94G86.1P01014.1 P22895.1 P43217.1 P55958.1 B8PYF3.1 O75475.1 O24554.1 Q0IX90.1Q52PJ2.1 K7VAC2.1 Q3Y8M6.1 Q9URR2.1 Q9P8G3.1 A1KYZ2.1 P23284.1 Q9TZR6.1Q25641.1 P00433.1 Q41260.1 P56164.1 Q40967.1 Q8H6L7.1 P35079.1 Q9XG86.1P43214.1 Q5ZQK5.1 Q40960.1 P43215.1 O82040.1 Q8L5D8.1 P82242.1 Q9HCM2.1Q9ZP03.1 Q9FPR0.1 B6T2Z8.1 Q9C5M8.1 P15722.1 P25788.1 P81651.1 O24248.1P82534.1 E3SH28.1 O65457.1 B6RQS1.1 P02761.1 P67876.1 Q9Y4W2.1 Q9ULX3.1P83181.1 Q8L5K9.1 C1KEU0.1 Q91482.1 Q9XHP1.1 P15322.1 Q15020.1 B9SA35.1P01267.1 O00267.1 D2T2K3.1 Q9T0P1.1 Q07283.1 Q7M3Y8.1 P25445.1 Q5NT95.1P07101.1 O15205.1 O00762.1 D2KFG9.1 H9AXB3.1 Q8W3V4.1 P49370.1 Q05110.1Q9ULJ6.1 Q2VST0.1 ABL09307.1 ABL09312.1 AGC39172.1 AGC39173.1 AGC39174.1P00785.4 P85204.1 AGC39168.1 CAM31908.1 ABB77213.1 P83958.1 AGC39176.1CAA34486.1 AAA32629.1 A5HII1.1 CAM31909.1 P85206.1 P86137.2 P85524.1CAI38795.2 ABQ42566.1 AAR92223.1 P84527.1 AGC39164.1 AGC39165.1AGC39166.1 AGC39167.1 4X9U_B AGC39169.1 AGC39170.1 AGC39171.1 AAC37218.1P50635.2 XP_001657556.2 P18153.2 AAB58417.1 ABF18122.1 XP_ XP_001653462.1 001654143.1 XP_001654291.1 ABF18258.1 XP_001655948.1XP_001655954.1 P13080.1 E37396 Q7M1X7 Q7M1X9 AAB24432.1 CAA76831.1AAB47552.1 AAM77471.1 AAS75297.1 3V0R_A 4AUD_B CAA55071.2 P49148.1Q6R4B4.1 P78983.2 Q00002.2 AAB48041.1 P42037.1 Q9HDT3.2 P42058.1OWY50380.1 AAO91800.1 P0C0Y4.2 AGS80276.1 CAD38167.1 ABI26088.1ACP43298.1 AKV72168.1 P27759.1 P27760.1 P27761.1 P28744.1 AAA32669.1CBW30986.1 CBW30987.1 CBW30988.1 CBW30989.1 CBW30990.1 CBW30991.1CBW30992.1 CBW30993.1 CBW30994.1 CBW30995.1 AAX77686.1 P27762.1CBJ24286.1 CBK52317.1 CBK62693.1 CBK62694.1 CBK62695.1 CBK62697.1CBK62698.1 CBK62699.1 O04004.1 AAP15203.1 AAP15202.1 AAP15201.1AAX77687.1 AAX77688.1 5EM1_A 5EV0_B AAX77684.1 AAX77685.1 AHA56102.15EGW_B P00304.2 P02878.1 AAA20065.1 AAA20067.1 AAA20064.1 AAA20066.1AAA20068.1 P10414.2 AEK65120.1 AAM73729.1 AAM73730.2 AAN76862.1AAL91665.1 O23791.1 Q94JN2.1 CDZ09832.1 AGC60026.1 AGC60027.1 AGC60028.1AGC60020.1 Q7Z1K3.1 AGC60035.1 AGC60036.1 ACZ95445.1 BAJ78220.1BAJ78221.1 BAJ78222.1 BAJ78223.1 AGC60029.1 AGC60030.1 AGC60031.1BAT62430.1 AAF75225.1 Q9NJA9.1 Q9NAS5.1 AEQ28167.1 P83885.1 CAK50389.1BAF43534.1 ABL77410.1 BAF75681.1 BAF75704.1 BAF75705.1 BAF75706.1BAF75707.1 BAF75708.1 BAF75709.1 BAF75710.1 BAF75711.1 BAF75712.1ABV55106.1 CAB58171.1 G37396 Q7M1X6 Q7M1Y0 A59055 AAK09361.1 Q7M4I5.1P01502.1 P00630.3 ABF21077.1 ABF21078.1 Q08169.1 ACI25605.1 Q5BLY5.1CAA26038.1 MEHB2 NP_001119715.1 NP_001035360.1 ABD51779.1 NP_001011564.1AAY21180.1 CAD56944.1 AHM25038.1 AHM25037.1 AHM25036.1 AHM25035.1P49372.1 P92918.1 ACV04796.1 AAD29409.1 P81943.3 P86809.1 AAB22817.1P43237.1 P43238.1 AAT00595.1 AAT00594.1 AAT00596.1 ADQ53858.1 3SMH_A3S7E_A B3EWP3.1 C0HJZ1.1 B3EWP4.1 AAN77576.1 AAM78596.1 AAK96887.1ACN62248.1 AAC63045.1 AAD47382.1 AAM46958.1 AAM93157.1 ABI17154.1ACH91862.1 3C3V_A ADQ53859.1 AAD55587.1 ADB96066.1 AGA84056.1 AAD56337.1AAL37561.1 1W2Q_A Q647G9.1 AAD56719.1 ABW17159.1 AAQ91847.1 ABP97433.1ACA79908.1 ABG85155.1 ABX56711.1 ABX75045.1 AAU21499.2 AAU21500.1AAZ20276.1 Q45W86 CAG26895.1 2X45_A AHF71021.1 AHF71022.1 AHF71023.1AHF71024.1 AHF71025.1 AHF71026.1 AAO24900.1 CAK50834.1 P0C088.1ACE07186.1 ACE07187.1 ACE07188.1 ACE07189.1 CAD12861.1 CAD12862.1 5EM0_AAAX85388.1 AAX85389.1 CAD23611.1 CAD23613.1 CAD23614.1 BAH09387.1AAD13644.1 AAD13645.1 AAD13647.1 AAD13649.1 AAD13650.1 AAD13651.1AAD13652.1 AAB93837.1 AAB93839.1 AAD13646.1 ACN32322.1 AAB26195.1Q06811.2 2XV9_A P46436.3 Q9UVU3 CAA06305.1 AAF86369.1 P67875.1CAA59419.1 CAB44442.1 CAA73782.1 AAB07620.1 P79017.2 AAK49451.1 Q96X30.3AAM43909.1 Q8NKF4.2 CAI78448.1 CAI78449.1 CAI78450.1 AAB95638.1CAM54066.1 CAA04959.1 O60024.2 CAA83015.1 P46075.3 AAB60779.1 Q92450.3O42799.2 CAB64688.1 Q9UUZ6.2 CAA11266.1 Q87519.1 EAL89830.1 Q4WB37.1KEY81716.1 KEY78748.1 AAA32702.1 CAB06417.1 AAD13106.1 P0C1B3.1AAA32708.1 P12547.2 ADE74975.1 P29600.1 P00780.1 AAG31026.1 BAA05540.1BAF46896.1 AIV43661.1 BAH10149.1 P04403.2 AAO38859.1 A45786 CAA54696.1CAA54695.1 CAA54694.1 CAA96546.1 CAA96539.1 CAA96540.1 CAA96541.1CAA96542.1 CAA96543.1 CAA96544.1 CAA96547.1 P43186.2 CAB02155.1CAB02156.1 CAB02157.1 CAB02158.1 CAB02159.1 CAB02160.1 CAB02161.1CAA96545.1 CAA05186.1 CAA05187.1 CAA05188.1 CAA05190.1 CAA07318.1CAA07319.1 CAA07323.1 CAA07324.1 CAA07325.1 CAA07326.1 CAA07327.1CAA07329.1 CAA07330.1 CAA04823.1 CAA04826.1 CAA04827.1 CAA04828.1CAA04829.1 AAD26560.1 AAD26561.1 AAD26562.1 P43180.2 1QMR_A AAP37482.11LLT_A AAB20452.1 CAA07328.1 CAA07320.1 CAA54488.1 1B6F_A 4BK7_A 4B9R_A4BKC_A 4BKD_A 4BK6_B CAA33887.1 CAA54482.1 CAA54483.1 CAA54484.1CAA54487.1 CAA54489.1 CAA54421.1 CAA54481.1 4BTZ_A 4Z3L_D B45786 1CQA_AAAA16522.1 A4K9Z8.1 CAA55854.1 CAA60628.1 AAG22740.1 CAC84116.1AHF71027.1 BAB21489.1 BAB21490.1 BAB21491.1 AAB25850.1 AAB25851.1AJO53282.1 AAB29344.1 AAB29345.1 ACM24358.1 ABC86902.1 AAD13531.1AAD13530.2 ABC68516.1 1YG9_A ABP35603.1 AAA86744.1 3LIZ_A ACY40650.1ACY40651.1 AAA87851.1 ABP04043.1 ACJ37389.1 ACF53836.1 ACF53837.1ABP04044.1 AAB72147.1 ABB89296.1 ABB89297.1 ABB89298.1 AAF72534.1ABX57814.1 AAK58415.1 AAQ24541.1 ABU97466.1 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ABX26147.1 ABX54842.1 ABX54844.1 ABX54849.1ABX54855.1 ABX54859.1 ABX54862.1 ABX54864.1 ABX54866.1 ABX54869.1ABX54876.1 ABX54877.1 AAB66909.1 P81430.2 AAF31152.1 AAF31151.1AAK58515.1 2JON_A BAE54432.1 Q25632.1 BAJ07603.1 P86431.1 P86432.1BAF95206.1 AFV53352.1 AAG42806.1 AAG42802.1 Q948T6.2 AAA86533.1AAF72991.1 BAB71741.1 Q40638.2 BAD13150.1 BAC20657.1 BAA01998.1BAA01996.1 BAA07772.1 BAA07773.1 BAA07774.1 BAA07710.1 BAA07711.1BAA07712.1 BAA07713.1 AAB99797.1 Q01882.2 Q01883.2 BAC19997.1 BAC20650.1ADK39021.1 ACA96507.1 CBY17558.1 AAC38996.1 BAF47265.1 BAF47266.12008179A CAA65123.1 CAA54587.1 CAI94601.1 CAA59370.1 CAA65122.1 P55958.1Q9T0M8.1 Q9XG85.1 CCP19647.1 CAP05019.1 Q7M1E8 AAB36008.1 AAB36009.1AAB36010.1 AAB36011.1 AAB36012.1 AAB46820.1 AAB46819.1 AKF12278.1CBM42667.1 CBM42666.1 CBM42665.1 CBM42664.1 CBM42663.1 CBM42662.1CBM42661.1 CBM42660.1 ACA23876.1 AAX37288.1 AAO15713.1 C7E3T4.1ADV17342.1 ADV17343.1 AAX11194.1 AAF71379.1 AAG44693.2 AAF23726.1AAM33821.1 AAB34785.1 ADK27483.1 AAD25995.1 AAG44480.1 Q92260.1AAK51201.1 AAR17475.1 AAD42074.1 ABB89950.1 ABM60783.1 AAD25926.1AEX34122.1 AAG44478.1 AKH04310.1 AKH04311.1 AAX33729.1 AEV23867.1AAD19606.1 CAB38086.1 ACS14052.1 AAC34736.1 AAC34737.1 AAB82404.1AAC34312.1 AAD13533.1 AAP13554.1 ADB92492.1 AAX33734.1 AAX33727.1ADR82198.1 AAB09632.1 AAB62731.1 AAB63595.1 Q25641.1 ADB92493.1ADD17628.1 AAX33728.1 3EBW_A ACJ37391.1 AAX33730.1 AAT77152.1 ACA00204.1AAL86701.1 AAG08988.1 CAB01591.1 AAB27445.1 Q41260.1 P56164.1 P56165.1P56166.1 P56167.1 ADC80502.1 ADC80503.1 CAA55390.1 CAA81613.1 1N10_ACAG24374.1 2118271A AAN32987.1 CAA70609.1 ABG81289.1 ABG81290.1ABG81291.1 ABG81292.1 ABG81293.1 ABG81294.1 ABG81295.1 CAA70608.1CAA54686.1 CAB42886.1 CAA53529.1 CAD54670.2 CAF32567.2 CAF32566.2CAQ55938.1 CAQ55939.1 CAQ55940.1 CAQ55941.1 3TSH_A CAD54671.2 CAA52753.1S32101 S38584 Q7M1L8 2023228A CAB05371.1 CAB05372.1 CAA50281.1AAC16525.1 AAC16526.1 AAC16527.1 AAC16528.1 AAC25994.1 AAC25995.1AAC25997.1 AAC25998.1 AAK25823.1 CAD38384.1 CAD38385.1 CAD38386.1CAD38387.1 CAD38388.1 CAD38389.1 CAD38390.1 CAD38391.1 CAD38392.1CAD38393.1 CAD38394.1 CAD38395.1 CAD38396.1 CAD38397.1 1L39_A CAD87529.1CAA81609.1 CCD28287.1 CAA76556.1 CAA76557.1 CAA76558.1 1NLX_N CAA76887.13FT1_A AGT28425.1 CAD10390.1 AHC94918.1 CEJ95862.1 CTQ87571.1 ABU42022.1ABG73109.1 ABG73110.1 ABG73108.1 ABO36677.1 ABR29644.1 CAF25233.1CAF25232.1 CAB82855.1 AJG44053.1 A0A158V755.1 A0A158V976.1 2N81_ACAC41633.1 CAC41634.1 CAC41635.1 CAD80019.1 ABY21305.1 ABY21306.1ALF39466.1 ALF00099.1 CAD20556.1 CAE52833.1 CAC85911.1 CBW45298.1 A60372F37396 CAA10520.1 AAG42254.1 P22284.1 P22286.1 A60373 P22285.1AAA29793.1 AAD52615.1 AAD52616.1 AAT95010.1 AAS67044.1 AAS67043.1AAS67042.1 AAS67041.1 AAP37412.1 AAT95009.1 P35780.1 P83377.1 P83542.1A2VBC4.1 ADT89774.1 ADL09135.1 P86687.1 ADD63684.1 P86686.1 Q7Z156.2P05946.1 AGE44125.1 ABL89183.1 ABS12234.1 AFA45339.1 ACN87223.1AKV72167.1 AHY24177.1 BAH59276.1 AAB97141.1 ADR66945.1 ADR66946.1ADR66947.1 ADR66948.1 AAC02632.1 AAS47037.1 AAS47036.1 AAS47035.1 1H2O_AAAF26449.1 ADR66943.1 ADR66944.1 AAD29411.1 AAB38064.1 P82534.1ACE80974.1 AAL91662.1 3EHK_A AGR27935.1 ADN39440.1 ADN39441.1 P82952.1ACE80939.1 ACE80956.1 ACE80958.1 ACE80957.1 ACE80959.1 ACE80955.1ACE80972.1 P83332.1 P83335.1 AEV57471.1 ABB78006.1 AJE61291.1 AJE61290.1P81402.1 AAV40850.1 ADR66939.1 AGW21344.1 CAD37201.1 CAD37202.1 P86888.1BAH10154.1 C0HKC0.1 AHB19227.1 AHB19226.1 AHB19225.1 AAF26451.1AET05733.1 AET05732.1 AET05730.1 O65200.1 AAD29410.1 AAC24001.1ABZ81045.1 ABZ81047.1 ABZ81046.1 CAC83046.1 CAC95152.1 CAC83047.1CAC95153.1 P02761.1 Q63213 AAA41198.1 AIS82657.1 AAP30720.1 AAT37679.1CAA38097.1 ABG54495.1 ABG54494.1 Q91483.3 ACI68103.1 CAA66403.1CBL79146.1 ACH70931.1 CBL79147.1 NP_001133181.1 AHL24657.1 ARS33724.1AAT99258.1 AAX11261.1 AAX11262.1 ACO34813.1 P83181.1 ACO34814.1ACS34771.1 AHL24658.1 ADK22841.1 ADK22842.1 CAX32966.1 CAX32967.1SHD75397.1 AAO15613.1 AAS93669.1 AAS93674.1 AAS93675.1 AAS93676.1AAO15607.1 AAX37321.1 AGM48615.1 CAQ68366.1 BAH10151.1 Q7M1Y1 C37396D37396 AAP06493.1 AAC67308.1 XP_003030591.1 BAW32538.1 BAW32537.1BAW32536.1 BAW32535.1 BAC66618.1 CAX32965.1 AFA45340.1 AFJ80778.1ABS12233.1 CAQ72968.1 CAQ72969.1 AAB37403.1 AAB37406.1 AAB34365.1CAH92630.1 CAH92627.1 Q7M263 CBG76811.1 BAE54429.1 BAE54430.1 ACB55491.1AAK15088.1 ACI41244.1 AAD42943.1 AAK15089.1 AAG23840.1 ACH85188.1AAD42942.1 AAD42944.1 AAK15087.1 CAA62909.1 CAA62910.1 CAA62911.1CAA62912.1 CAA62908.1 P15322.2 AAX77383.1 AAX77384.1 ABU95411.1ABU95412.1 ABU53681.1 NP_ 001306883.1 NP_001316123.1 CAD10377.1AAL29690.1 AAL75449.1 AAL75450.1 CAJ19705.1 AAB42069.1 CAA75803.1AHC08074.1 AHC08073.1 ABA81885.1 ABB16985.1 CAA31575.1 CAA27571.1CAA27588.1 AAA33819.1 P15476.2 P16348.1 P20347.3 AAB63099.1 BAA04149.1BAH10156.1 AAF65312.1 AAF65313.1 AAC97370.1 AAC97369.1 AAB36117.1AAB36119.1 AAB36120.1 AAB36121.1 AAT95008.1 P35775.1 AAB65434.1 P35776.2P35779.2 ADD74392.1 AIL01319.1 AIL01318.1 AIL01316.1 AIL01317.1AIL01320.1 AIL01321.1 ACT37324.1 1ESF_B CAJ43561.1 P34071.1 P20723.1P06886.1 AAT66567.1 ABS29033.1 AAT66566.1 AAD46493.1 AAS75831.1 P00791.3AAA30988.1 NP_ 001005208.1 P58171.1 S43242 S43243 S43244 ADX78255.1ADM18346.1 ADM18345.1 ADK47876.1 P86360.1 CEE03319.1 CEE03318.1AAK63089.1 AAK63088.1 CBL79145.1 P86978.1 CAX62602.1 P86979.1 BAE54431.1BAE46763.1 BAH10155.1 AAF07903.2 AAD52013.1 AAD52012.1 Q8J077.1CAD23374.1 P24296.2 CAA42453.1 ACG59281.1 AKJ77988.1 AKJ77986.1AKJ77987.1 CAI64398.1 AKJ77990.1 AKJ77985.1 CAA35238.1 CAA25593.1CAA26383.1 CAA26384.1 CAA26385.1 AAA34275.1 AAA34276.1 AAA34279.1AAA34280.1 AAA34281.1 AAA34282.1 AAA34283.1 AAA34284.1 BAA12318.1P81496.1 ACE82289.1 BAE20328.1 CAR82265.1 CAR82266.1 CAR82267.1BAN29067.1 CAI64397.1 CAI64396.1 P08819.2 P27357.1 ACE82291.1 CAA61945.2CAA61943.2 CAA61944.2 CAQ57979.1 CBA13560.1 AAA34272.1 AAA34274.1AAA34288.1 AAA34289.1 BAA11251.1 CAI78902.1 BAN29066.1 CAY54134.1CAB96931.1 CAA43331.1 CAA31396.1 CAA26847.1 CAA24934.1 CAA43361.1AAB02788.1 CAA27052.1 CAA24933.1 BAN29068.1 CAA31395.4 AAZ23584.1BAC76688.1 CAI84642.1 CAA35598.1 CAZ76052.1 CBA13559.1 CAA35597.1CAC14917.1 ACE82290.1 Q6W8Q2.1 CAA72273.1 CAB52710.1 CAZ76054.1CAA31685.1 CAA30570.1 AAA34285.1 AAA34286.1 AAA34287.1 O22116 CAA59338.1CAA59339.1 CAA59340.1 O22108 CAI79052.1 AEH31546.1 BAN29069.1 CAA65313.1ABS58503.1 P82977.2 CCK33471.1 APY24042.1 CAA34709.1 CAA39099.1CAA36063.1 CAA44473.1 AAA34290.1 AAX34057.1 AAX34058.1 AAX34059.1AOD75395.1 AOD75396.1 AOD75399.1 ABQ96644.1 ABU97479.1 AAT40866.1AAU11502.1 ABM53751.1 ABU97480.1 CAA73221.1 ACL36923.1 ABZ81991.1AGG10560.1 AAT66607.1 AAT66609.1 ACH42744.1 AAT66610.1 ACJ65836.1AGC36415.1 ACH42743.1 ACI44002.1 ABQ59259.1 ABQ59258.1 ABQ59255.1ACJ54737.1 ACH42741.1 AGC36416.1 AKV72166.1 AIV43662.1 BAH10157.1P0DMB5.1 P0DMB4.1 P0CH87.1 P35781.1 P35782.1 CBY83816.1 CBY93636.1P81657.1 P35783.1 CAJ28931.1 P35784.1 CAJ28930.1 CAL59818.1 CAL59819.1P51528.1 P35760.1 ABC73068.1 P0CH89.1 P35785.1 P35786.1 P0CH86.1P35787.1 AAB48072.1 AAA30333.1 CAB42887.1 1QNX_A P49370.1 CAI77218.12ATM_A ACA00159.1 AAX19889.1 ABG02262.1 ABW23574.1 BAA74451.1 CAA50008.1P80273.2 P80274.1 P33556.1 CAR48256.1 ABD79096.1 ABD79097.1 ABD79098.1ACX37090.1 P29022.1 2209273A AAO45607.1 AAO45608.1 AAK56124.1 2HCZ_XABD79094.1 ABD79095.1 ABF81661.1 ABF81662.1 Q1ZYQ8.2 P0C1Y5.1 AAB86960.1ABG81312.1 ABG81313.1 ABG81314.1 ABG81315.1 ABG81316.1 ABG81317.1ABG81318.1 CAA51718.1 CAA51719.1 CAA51720.1 AAG35601.1 5FEF_A AAA33493.1AAA33494.1 CAI64400.1 AAX40948.1

TABLE 7 LIST OF ACCESSION NUMBERS FOR AUTOMIMMUNE ANTIGENS FROM IEDBI7HKY1.1 Q9P0J1.1 P61604.1 Q9NUQ2.1 Q9P212.1 P16885.1 P09543.1 P17980.1Q99460.1 O00231.1 O00487.1 P48556.1 Q61733.1 P82909.1 P21953.1 Q9CHK3.1Q9BYD6.1 Q9BYC9.1 Q96A35.1 Q9P0J6.1 P04035.1 Q99714.1 B2RLH8.1 P62277.1P08708.1 P62269.1 P63220.1 P62851.1 P62273.1 P62861.1 P46781.1 P08865.1P17643.1 Q9H0D6.1 F5HCM1.1 E5RK45.1 A0A0B7JKK9.1 A1JIP3.1 B2RKS6.1P0A6F5.1 P0C0Z7.1 Q49375.1 Q9Z708.1 P0A521.1 P42384.1 P0A520.1 P9WPE7.1P10809.1 P10155.1 P05388.1 P05386.1 P05387.1 P27635.1 P62906.1 P40429.1P35268.1 A8MUS3.1 P62750.1 P61353.1 P46776.1 P46779.1 P47914.1 P39023.1P62888.1 Q02878.1 P18124.1 P62917.1 P32969.1 Q6SW59.1 P08253.1 P11021.1Q969T7.1 Q76LX8.1 C6AV76.1 Q2FWL5.1 B1RDC1.1 Q2G2D8.1 P42684.1 Q8IZT6.1Q9Y4K1.1 P02709.1 P02710.1 P02711.1 P04756.1 P02708.1 P02712.1 P11230.1Q07001.1 P02715.1 Q04844.1 P07510.1 P13536.1 F1N690.1 M9YGB9.1 O43427.1P68133.1 P62736.1 P60709.1 P63261.1 Q9NQW6.1 O15144.1 Q9H981.1 Q8N3C0.1Q6VMQ6.1 Q6JQN1.1 Q5T8D3.1 P82987.1 Q6ZMM2.1 Q9NZK5.1 Q8IUX7.1 Q9NP61.1Q9UJY4.1 O43488.1 P07897.1 P16112.1 Q73ZL3.1 Q92667.1 P49588.1 C9JKR2.1F8ELD9.1 P15121.1 F5HF49.1 P05186.1 P55008.1 Q5STX8.1 P02763.1 P01009.1P35368.1 P04217.1 P25100.1 P08697.1 P18825.1 P02765.1 P01023.1 P12814.1O43707.1 P35611.1 Q9UBT7.1 P61163.1 P02489.1 P02511.1 P06733.1 P06280.1Q16352.1 Q96Q83.1 P37840.1 Q9UJX4.1 P01019.1 Q9P2G1.1 Q9H8Y5.1 Q8N6D5.1H0YKS4.1 P04083.1 P50995.1 P07355.1 P08758.1 P08133.1 Q9NQ90.1 Q03518.1P01008.1 Q10567.1 Q9BXS5.1 Q96CW1.1 O00203.1 P02647.1 P02652.1 P06727.1P04114.1 P02655.1 C9JX71.1 P05090.1 P02649.1 Q9BZR8.1 P03182.1 Q9BRQ8.1Q9ATL6.1 P47863.1 P55087.1 P55064.1 P20292.1 Q15057.1 Q96P48.1 P35869.1Q5VUY2.1 P03928.1 P25705.1 P06576.1 P56385.1 Q9DB20.1 P18859.1 Q9BZC7.1Q8WWZ7.1 Q9NUT2.1 P61221.1 P53396.1 A1JNN2.1 P0A6G7.1 Q9H2U1.1 Q14562.1O84848.1 P78508.1 Q99712.1 P17342.1 Q99856.1 Q81VW6.1 Q96GD4.1 Q8WXX7.1O15392.1 P02730.1 P98160.1 F8W034.1 P20749.1 P41182.1 Q9NYF8.1 Q6W2J9.1Q8NFU0.1 P15291.1 P07550.1 P02749.1 P61769.1 Q13425.1 Q562R1.1 P42025.1P13929.1 F0K2P6.1 O43252.1 Q13057.1 Q81UF8.1 Q8NFC6.1 P18577.1 Q5VSJ8.1Q02161.1 P02663.1 P02769.1 Q9NWK9.1 O95415.1 Q7Z569.1 Q99728.1 Q9P287.1Q9NRL2.1 Q9UIF9.1 Q58F21.1 P25440.1 Q15059.1 O60885.1 P18892.1 Q8NCU7.1P04003.1 O75844.1 P12830.1 P33151.1 Q8NE86.1 P62158.1 P07384.1 P17655.1P20810.1 P27797.1 O94985.1 P10644.1 P31321.1 P13861.1 O70739.1 Q8QVL3.1Q8QVL6.1 Q8QVL9.1 Q91CY5.1 Q91CZ6.1 Q98Y63.1 Q99AQ9.1 Q9DTD4.1 Q9DUB7.1Q9DUC1.1 Q9JG76.1 Q9QU30.1 Q9QUB8.1 Q80AR5.1 Q80QT8.1 Q8UZK7.1 P14348.1Q9H2A9.1 P00918.1 P16870.1 O75339.1 O15519.1 Q14790.1 P04040.1 P35221.1P49913.1 P07858.1 P07339.1 P25774.1 Q03135.1 Q16663.1 Q9H9A5.1 Q9Y5K6.1P09326.1 P14209.1 Q99741.1 O00311.1 O75794.1 P04637.1 B2RD01.1 Q03188.1P49454.1 Q9HC77.1 Q02224.1 P00450.1 P08622.1 P35514.1 Q05980.1 P9WMJ9.1Q9H444.1 P36222.1 O00299.1 P05108.1 O15335.1 Q6UVK1.1 Q9P2D1.1 P10645.1O75390.1 O14503.1 Q00610.1 P09497.1 O75508.1 P56750.1 Q9P210.1 Q7Z460.1O75122.1 O75153.1 P10909.1 Q7Z401.1 P00451.1 P00488.1 P48444.1 P61923.1E9PP50.1 P23528.1 Q8WUD4.1 Q49A88.1 Q16204.1 P38432.1 P02452.1 P02458.1P05539.1 P02462.1 G1K238.1 Q7SIB2.1 P20908.1 Q02388.1 P27658.1 P12107.1Q99715.1 Q05707.1 P39059.1 Q9UMD9.1 P08123.1 P08572.1 Q7SIB3.1 P05997.1P12110.1 P13942.1 F1MZU6.1 Q01955.1 P12111.1 P02745.1 P02746.1 P09871.1P01024.1 P0C0L5.1 P01031.1 Q07021.1 P13671.1 P02748.1 P08603.1 Q03591.1Q6PUV4.1 W1Q7Z5.1 Q15021.1 Q15003.1 P42695.1 Q14746.1 Q9NZB2.1 Q12860.1Q02246.1 P78357.1 Q9UBW8.1 P36717.1 P02741.1 P12277.1 P06732.1 H0Y8U5.1Q13618.1 Q86VP6.1 P25024.1 P16220.1 P06493.1 P11802.1 Q00534.1 P50750.1P41002.1 P04080.1 P50238.1 P52943.1 O14957.1 P20674.1 P10606.1 P14854.1P15954.1 P10176.1 Q16678.1 P10635.1 Q14008.1 Q9Y5Y2.1 Q96KP4.1 P14416.1Q5QP82.1 P07585.1 E5RFJ0.1 Q86SQ9.1 Q9Y394.1 P49366.1 Q5QJE6.1 P24855.1Q02413.1 P32926.1 P15924.1 Q16760.1 P19572.1 A9NHS5.1 Q9JZ09.1 P06959.1P08461.1 P10515.1 P20285.1 P0AFG6.1 Q5F875.1 P19262.1 P36957.1 Q16555.1P53634.1 Q14689.1 Q13443.1 Q12959.1 Q15398.1 Q16531.1 P40692.1 P43246.1P09884.1 P03198.1 P04293.1 Q9NRF9.1 Q9UGP5.1 P89471.1 Q13426.1 P49736.1P33992.1 P11387.1 Q02880.1 Q9UBZ4.1 P24928.1 O14802.1 Q9NW08.1 P31689.1P25686.1 O60216.1 O95793.1 P55265.1 Q6P0N6.1 Q13202.1 Q8IVF4.1 E9PEB9.1Q9UII4.1 P11161.1 Q14258.1 Q9ULT8.1 O95714.1 Q7Z6Z7.1 Q9Y4L5.1 O43567.1Q63HN8.1 Q969K3.1 Q8IUQ4.1 P19474.1 Q6AZZ1.1 Q9C026.1 Q14669.1 Q5T4S7.1P18146.1 Q05BV3.1 Q6ZMW3.1 O95967.1 P15502.1 Q9BY07.1 P13804.1 Q6PJG2.1A6PW80.1 P68104.1 P13639.1 Q96RP9.1 Q9BW60.1 Q9UI08.1 P17813.1 Q9NZ08.1P14625.1 Q14511.1 Q6P2E9.1 B2RLL7.1 O84591.1 Q9Z7A6.1 P03188.1 P04578.1P14075.1 Q6SW67.1 Q92817.1 P12724.1 Q12929.1 P61916.1 P07099.1 P03211.1P12978.1 P12977.1 P03203.1 P03204.1 Q99808.1 P27105.1 P03372.1 P32519.1Q15723.1 P60842.1 Q14240.1 P38919.1 P41567.1 Q14152.1 B5ME19.1 P60228.1O75821.1 Q13347.1 Q9Y262.1 F1TIN3.1 Q96KP1.1 Q96A65.1 O84646.1 Q01780.1P30822.1 O14980.1 P41180.1 P15311.1 Q08945.1 P52907.1 Q9BXW9.1 Q14296.1Q16658.1 Q7L8L6.1 Q7L5A8.1 P49327.1 Q8IX29.1 Q8TB52.1 Q7Z6M2.1 Q71513.1Q9BZ67.1 A1ZL39.1 P02792.1 P35555.1 P02671.1 P02675.1 P02679.1 Q06828.1P02751.1 Q4ZHG4.1 P20930.1 P21333.1 P30043.1 O75955.1 Q14254.1 P49771.1Q12841.1 Q13461.1 P32314.1 O95954.1 P04075.1 P09972.1 P07954.1 Q9H0Q3.1Q7Z6J4.1 P30279.1 P30281.1 O96020.1 O95067.1 P14078.1 P51570.1 Q08380.1O00214.1 Q3B8N2.1 P34903.1 P09104.1 A4D1B5.1 P17900.1 P06396.1 Q12789.1Q8WUA4.1 P03300.1 P08292.1 P27958.1 P03995.1 P14136.1 P47871.1 Q8TDQ7.1P35575.1 Q9NQR9.1 Q9Z186.1 P11413.1 P06744.1 P48318.1 Q99259.1 P48320.1Q05329.1 Q05683.1 P00367.1 Q05586.1 Q5VSF9.1 Q12879.1 S0G235.1 P15104.1Q06210.1 P35754.1 P18283.1 P09211.1 P04406.1 Q9NPB8.1 P11216.1 P06737.1P11217.1 Q31BS5.1 P04921.1 O43292.1 P30419.1 D6RB28.1 Q96S52.1 Q969N2.1Q86SQ4.1 Q9HC97.1 K7EQ05.1 P28799.1 P0A6P5.1 P44536.1 Q8WWP7.1 P62826.1P16520.1 P09471.1 Q9BVP2.1 Q9NVN8.1 P00738.1 Q9Y6N9.1 Q96CS2.1 P48723.1Q0VDF9.1 P08107.1 P34931.1 P11142.1 P04792.1 P07900.1 Q14568.1 P08238.1P54652.1 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Predicting the Immunological Response of an Individual to a PolypeptideAntigen

Specific polypeptide antigens induce immune responses in only a fractionof human subjects. Currently, there is no diagnostic test that canpredict whether a polypeptide antigen would likely induce an immuneresponse in an individual. In particular, there is a need for a testthat can predict whether a person is an immune responder to a vaccine orimmunotherapy composition.

According to the present disclosure, the polypeptide antigen-specific Tcell response of an individual is defined by the presence within thepolypeptide of one or more fragments that may be presented by multipleHLA class I or multiple HLA class II molecules of the individual.

In some cases the disclosure provides a method of predicting whether asubject will have an immune response to administration of a polypeptide,wherein an immune response is predicted if the polypeptide isimmunogenic according to any method described herein. A cytotoxic T cellresponse is predicted if the polypeptide comprises at least one aminoacid sequence that is a T cell epitope capable of binding to at leasttwo HLA class I molecules of the subject. A helper T cell response ispredicted if the polypeptide comprises at least one amino acid sequencethat is a T cell epitope capable of binding to at least two HLA class IImolecules of the subject. No cytotoxic T cell response response ispredicted if the polypeptide does not comprise any amino acid sequencethat is a T cell epitope capable of binding to at least two HLA class Imolecules of the subject. No helper T cell response is predicted if thepolypeptide does not comprise any amino acid sequence that is a T cellepitope capable of binding to at least two HLA class II molecules of thesubject.

In some cases the polypeptide is an active component of a pharmaceuticalcomposition, and the method comprises predicting the development orproduction of anti-drug antibodies (ADA) to the polypeptide. Thepharmaceutical composition may be a drug selected from those listed inTable 8. According to the present disclosure, ADA development will occurif, or to the extent that, an active component polypeptide is recognisedby multiple HLA class II molecules of the subject, resulting in a helperT cell response to support an antibody response to the active component.The presence of such epitopes (PEPIs) may predict the development of ADAin the subject. The method may further comprise selecting orrecommending for treatment of the specific human subject administrationto the subject of a pharmaceutical composition that is predicted toinduce low or no ADA, and optionally further administering thecomposition to the subject. In other cases the method predicts that thepharmaceutical composition will induce unacceptable ADA and the methodfurther comprises selecting or recommending or treating the subject witha different treatment or therapy. The polypeptide may be a checkpointinhibitor. The method may comprise predicting whether the subject willrespond to treatment with the checkpoint inhibitor.

TABLE 8 Example drugs associated with ADA-related adverse events DrugADA-related adverse event Abciximab anaphylaxis Adalimumab anti-drugantibodies and treatment failure Basiliximab anaphylaxis Cetuximab IgE,anaphlyaxis Epoetin Antibody-mediated pure red cell aplasiaErythropoietin pure red cell aplasia Etanercept no apparent effect onsafety Factor-IX anaphylaxis Infliximab anaphylaxis OKT3 anaphylaxisPegloticase anti-dug antibody, treatment failure rIFN-beta anaphylaxisrecombinant factor VIII anaphylaxis Thrombopoietin thrombocitopeniaUstekinumab anti-ustekinumab antibodies, affected treatment efficacy

There is also currently no test that can predict the likelihood that aperson will have a clinical response to, or derive clinical benefitfrom, a vaccine or immunotherapy composition. This is important becausecurrently T cell responses measured in a cohort of individualsparticipating in vaccine or immunotherapy clinical trials poorlycorrelate with clinical responses. That is, the clinical respondersubpopulation is substantially smaller than the immune respondersubpopulation. Therefore, to enable the personalization of vaccines andimmunotherapies it is important to predict not only the likelihood of animmune response in a specific subject, but also whether the immuneresponse induced by the drug will be clinically effective (e.g. can killcancer cells or pathogen infected cells or pathogens).

The inventors have discovered that the presence in a vaccine orimmunotherapy composition of at least two polypeptide fragments(epitopes) that can bind to at least three HLA class I of an individual(≥2 PEPI3+) is predictive for a clinical response. In other words, if ≥2PEPI3+ can be identified within the active ingredient polypeptide(s) ofa vaccine or immunotherapy composition, then an individual is a likelyclinical responder. A “clinical response” or “clinical benefit” as usedherein may be the prevention of or a delay in the onset of a disease orcondition, the amelioration of one or more symptoms, the induction orprolonging of remission, or the delay of a relapse or recurrence ordeterioration, or any other improvement or stabilisation in the diseasestatus of a subject. Where appropriate, a “clinical response” maycorrelate to “disease control” or an “objective response” as defined bythe Response Evaluation Criteria In Solid Tumors (RECIST) guidelines.

Therefore, in some cases the disclosure provides a method of predictingwhether the subject will have a clinical response to administration of apharmaceutical composition such as a vaccine or immunotherapycomposition comprising one or more polypeptides as active ingredients.The method may comprise determining whether the one or more polypeptidestogether comprise at least two different sequences each of which is a Tcell epitope capable of binding to at least two, or in some cases atleast three HLA class I molecules of the subject; and predicting thatthe subject will have a clinical response to administration of thepharmaceutical composition if the one or more polypeptides togethercomprise at least two different sequences each of which is a T cellepitope capable of binding to at least two, or in some cases at leastthree HLA class I molecules of the subject; or that the subject will nothave a clinical response to administration of the pharmaceuticalcomposition if the one or more polypeptides together comprise no morethat one sequence that is a T cell epitope capable of binding to atleast two, or in some cases at least three HLA class I molecules of thesubject.

For the purposes of this method two T cell epitopes are “different” fromeach other if they have different sequences, and in some cases also ifthey have the same sequence that is repeated in a target polypeptideantigen. In some cases the different T cell epitopes in a targetpolypeptide antigen do not overlap with one another.

In some cases all of the fragments of one or more polypeptides or activeingredient polypeptides that are immunogenic for a specific humansubject are identified using the methods described herein. Theidentification of at least one fragment of the polypeptide(s) that is aT cell epitope capable of binding to at least two, or at least three HLAclass I molecules of the subject predicts that the polypeptide(s) willelicit or is likely to elicit a cytotoxic T cell response in thesubject. The identification of at least one fragment of thepolypeptide(s) that is a T cell epitope capable of binding to at leasttwo, or at least three, or at least four HLA class II molecules of thesubject predicts that the polypeptide(s) will elicit or is likely toelicit a helper T cell response in the subject. The identification of nofragments of the polypeptide(s) that are T cell epitopes capable ofbinding to at least two, or at least three HLA class I molecules of thesubject predicts that the polypeptide(s) will not elicit or is notlikely to elicit a cytotoxic T cell response in the subject. Theidentification of no fragments of the polypeptide(s) that are T cellepitopes capable of binding to at least two, or at least three, or atleast four HLA class II molecules of the subject predicts that thepolypeptide(s) will not elicit or is not likely to elicit a helper Tcell response in the subject. The identification of at least twofragments of one or more active ingredient polypeptides of a vaccine orimmunotherapy composition, wherein each fragment is a T cell epitopecapable of binding to at least two, or at least three HLA class Imolecules of the subject predicts that the subject is more likely tohave, or will have a clinical response to the composition. Theidentification of less than two fragments of the one or morepolypeptides that are T cell epitopes capable of binding to at leasttwo, or at least three HLA class I molecules of the subject predictsthat the subject is less likely to have, or will not have, a clinicalresponse to the composition.

Without wishing to be bound by theory, one reason for the increasedlikelihood of deriving clinical benefit from a vaccine/immunotherapycomprising at least two multiple-HLA binding PEPIs, is that diseasedcell populations, such as cancer or tumor cells or cells infected byviruses or pathogens such as HIV, are often heterogenous both within andbetween effected subjects. A specific cancer patient, for example, mayor may not express or overexpress a particular cancer associated targetpolypeptide antigen of a vaccine, or their cancer may compriseheterogeneous cell populations, some of which (over-)express the antigenand some of which do not. In addition, the likelihood of developingresistance is decreased when more multiple HLA-binding PEPIs areincluded or targeted by a vaccine/immunotherapy because a patient isless likely to develop resistance to the composition through mutation ofthe target PEPI(s).

The likelihood that a subject will respond to treatment is thereforeincreased by (i) the presence of more multiple HLA-binding PEPIs in theactive ingredient polypeptides; (ii) the presence of PEPIs in moretarget polypeptide antigens; and (iii) (over-)expression of the targetpolypeptide antigens in the subject or in diseased cells of the subject.In some cases expression of the target polypeptide antigens in thesubject may be known, for example if target polypeptide antigens are ina sample obtained from the subject. In other cases, the probability thata specific subject, or diseased cells of a specific subject,(over-)express a specific or any combination of target polypeptideantigens may be determined using population expression frequency data.The population expression frequency data may relate to a subject- and/ordisease-matched population or the intent-to-treat population. Forexample, the frequency or probability of expression of a particularcancer-associated antigen in a particular cancer or subject having aparticular cancer, for example breast cancer, can be determined bydetecting the antigen in tumor, e.g. breast cancer tumor samples. Insome cases such expression frequencies may be determined from publishedfigures and scientific publications. In some cases a method of theinvention comprises a step of determining the expression frequency of arelevant target polypeptide antigen in a relevant population.

Disclosed is a range of pharmacodynamic biomarkers to predict theactivity/effect of vaccines in individual human subjects as well as inpopulations of human subjects. The biomarkers have been developedspecifically for cancer vaccines, but similar biomarkers could be usedfor other vaccines or immunotherapy compositions. These biomarkersexpedite more effective vaccine development and also decrease thedevelopment cost and may be used to assess and compare differentcompositions. Exemplary biomarkers are as follows.

-   -   AG95—potency of a vaccine: The number of antigens in a cancer        vaccine that a specific tumor type expresses with 95%        probability. AG95 is an indicator of the vaccine's potency, and        is independent of the immunogenicity of the vaccine antigens.        AG95 is calculated from the tumor antigen expression rate data.        Such data may e obtained from experiments published in peer        reviewed scientific journals. Technically, AG95 is determined        from the binomial distribution of antigens in the vaccine, and        takes into account all possible variations and expression rates.    -   PEPI3+ count—immunogenicity of a vaccine in a subject:        Vaccine-derived PEPI3+ are personal epitopes that bind to et        least 3 HLAs of a subject and induce T cell responses. PEPI3+        can be determined using the PEPI3+ Test in subjects who's        complete 4-digit HLA genotype is known.    -   AP count—antigenicity of a vaccine in a subject: Number of        vaccine antigens with PEPI3+. Vaccines contain sequences from        target polypeptide antigens expressed by diseased cells. AP        count is the number of antigens in the vaccine that contain        PEPI3+, and the AP count represents the number of antigens in        the vaccine that can induce T cell responses in a subject. AP        count characterizes the vaccine-antigen specific T cell        responses of the subject since it depends only on the HLA        genotype of the subject and is independent of the subject's        disease, age, and medication. The correct value is between 0 (no        PEPI presented by the antigen) and maximum number of antigens        (all antigens present PEPIs).    -   AP50—antigenicity of a vaccine in a population: The mean number        of vaccine antigens with a PEPI in a population. The AP50 is        suitable for the characterization of vaccine-antigen specific T        cell responses in a given population since it depends on the HLA        genotype of subjects in a population.    -   AGP count—effectiveness of a vaccine in a subject: Number of        vaccine antigens expressed in the tumor with PEPI. The AGP count        indicates the number of tumor antigens that vaccine recognizes        and induces a T cell response against (hit the target). The AGP        count depends on the vaccine-antigen expression rate in the        subject's tumor and the HLA genotype of the subject. The correct        value is between 0 (no PEPI presented by expressed antigen) and        maximum number of antigens (all antigens are expressed and        present a PEPI).    -   AGP50—effectiveness of a cancer vaccine in a population: The        mean number of vaccine antigens expressed in the indicated tumor        with PEPI (i.e., AGP) in a population. The AGP50 indicates the        mean number of tumor antigens that the T cell responses induced        by the vaccine can recognize. AGP50 is dependent on the        expression rate of the antigens in the indicated tumor type and        the immunogenicity of the antigens in the target population.        AGP50 can estimate a vaccine's effectiveness in different        populations and can be used to compare different vaccines in the        same population. The computation of AGP50 is similar to that        used for AG50, except the expression is weighted by the        occurrence of the PEPI3+ in the subject on the expressed vaccine        antigens. In a theoretical population, where each subject has a        PEPI from each vaccine antigen, the AGP50 will be equal to AG50.        In another theoretical population, where no subject has a PEPI        from any vaccine antigen, the AGP50 will be 0. In general, the        following statement is valid: 0≤AGP50≤AG50.    -   mAGP—a candidate biomarker for the selection of likely        responders: Likelihood that a cancer vaccine induces T cell        responses against multiple antigens expressed in the indicated        tumor. mAGP is calculated from the expression rates of        vaccine-antigens in in e.g. the tumor and the presence of        vaccine derived PEPIs in the subject. Technically, based on the        AGP distribution, the mAGP is the sum of probabilities of the        multiple AGP (≥2 AGPs).

The results of a prediction as set out above may be used to inform aphysician's decisions concerning treatment of the subject. Accordingly,in some cases the polypeptide is an active ingredient, for example of avaccine or immunotherapy composition, the method of the diclosurepredicts that the subject will have, is likely to have, or has above athreshold minimum likelihood of having an immune response and/or aclinical response to a treatment comprising administering the activeingredient polypeptide to the subject, and the method further comprisesselecting the treatment for or selecting the vaccine or immunotherapycomposition for treatment of the specific human subject. Also providedis a method of treatment with a subject-specific pharmaceuticalcomposition, kit or panel of polypeptides comprising one or morepolypeptides as active ingredients, wherein the pharmaceuticalcomposition, kit or panel of polypeptides has been determined to have athreshold minimum likelihood of inducing a clinical response in thesubject, wherein the likelihood of response has been determined using amethod described herein. In some cases the minimum threshold is definedby one or more of the pharmacodynamic biomarkers described herein, forexample a minimum PEPI3+ count (for example 2, 3, 4, 5, 6, 7, 8, 9, 10,11, or 12 or more PEPI3+), a minimum AGP count (for example AGP=at least2 or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more) and/or aminimum mAGP (for example AGP=at least 2 or at least 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 or more). For example, in some cases a subject isselected for treatment if their likelihood of a response targeted at apredefined number of target polypeptide antigens, optionally wherein thetarget polypeptide antigens are (predicted to be) expressed, is above apredetermined threshold (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 ormore). Alternatively, the method may predict that the one or morepolypeptide(s) of the composition will not elicit a T cell responseand/or a clinical response in the subject and further comprise selectinga different treatment for the specific human subject.

Predicting an Autoimmune or Toxic Immune Response to a PolypeptideAntigen

The differences among HLAs may influence the probability of developingan autoimmune disease, condition or response. In some cases the methodof the disclosure may be used to identify a polypeptide or a fragment ofa polypeptide that is immunogenic and/or associated with an auto-immunedisorder or response. In some cases, the method comprises determiningwhether a polypeptide comprises an amino acid sequence that is a T cellepitope capable of binding to at least three, or at least four, or atleast five HLA class I of a subject; or in other cases a sequence thatis a T cell epitope capable of binding to at least four, or at leastfive, or at least six HLA class II of a subject; and identifying thepolypeptide or said sequence as immunogenic or as being related to orassociated with an auto-immune disorder or an auto-immune response inthe subject.

The differences among HLAs may also influence the probability that asubject will experience immune-toxicity from a drug or polypeptideadministered to the subject. There may be a toxic immune response if apolypeptide administered to the subject comprises a fragment thatcorresponds to a fragment of an antigen expressed in normal healthycells of the subject and that comprises an amino acid that is a T cellepitope capable of binding to multiple HLA class I molecules of thesubject. Therefore, in some cases in accordance with the disclosure, themethod is used to identify a toxic immunogenic region or fragment of apolypeptide or to identify subjects who are likely to experienceimmune-toxicity in response to administration of one or morepolypeptides or a fragments thereof. The polypeptide may be an activeingredient of a vaccine or immunotherapy composition.

The method may comprise determining whether the polypeptide(s) comprisesa sequence that is a T cell epitope capable of binding to at least two,or in other cases to at least three HLA class I molecules of thesubject. In some cases the method comprises determining that thepolypeptide comprises a sequence that is a T cell epitope capable ofbinding to at least four, or at least five HLA class I molecules of thesubject; or an amino acid sequence that is a T cell epitope capable ofbinding to at least four, or at least five, or at least six or at leastseven HLA class II of the subject. The method may further compriseidentifying said sequence as toxic immunogenic for the subject orpredicting a toxic immune response in the subject. In other cases nosuch amino acid sequence is identified and the method further comprisespredicting no toxic immune response in the subject. The method mayfurther comprise selecting or recommending for treatment of the subjectadministration of one or more polypeptides or a pharmaceuticalcomposition that is predicted to induce no or low immune-toxicity, andoptionally further treating the subject by administering thepolypeptide. The disclosure also provides a method of treating a subjectin need thereof by administrating to the subject such a polypeptide orcomposition.

In some cases a method described herein further comprises mutating apolypeptide that is predicted to be immunogenic for a specific humansubject, or that is predicted to be immunogenic in a proportion ofsubjects in a human population. Also provided is a method of reducingthe immunogenicity of a polypeptide that has been identified asimmunogenic in a specific human subject or in a proportion of a humanpopulation using any one of the methods described herein. Thepolypeptide may be mutated to reduce the number of PEPIs in thepolypeptide or to reduce the number of HLA class I or class II moleculesof the subject or of said population that bind to the fragment of thepolypeptide that is identified as immunogenic in the subject or in aproportion of said population. In some cases the mutation may reduce orprevent a toxic immune response or may increase the efficacy bypreventing the ADA development in the subject or in a proportion of saidpopulation. The mutated polypeptide may be further selected orrecommended for treatment of the subject or of a subject of saidpopulation. The subject may further be treated by administration of themutated polypeptide. The disclosure also provides a method of treating asubject in need thereof by administrating to the subject such a mutatedpolypeptide.

Predicting the Response of an Individual to Treatment with a CheckpointInhibitor

Typically some or all of the tumor specific T cell clones that areinduced by a tumor are inactive or poorly functional in metastaticcancer patients. Inactive tumor specific T cells cannot kill the tumorcells. A fraction of these inactive T cells may be re-activated bycheckpoint inhibitors (such as Ipilimumab), for example monoclonalantibodies that recognize checkpoint molecules (e.g. CTLA-4, PD-1,Lag-3, Tim-3, TIGIT, BTLA). According to the present disclosure,treating a subject with a checkpoint inhibitor will only be effective ifor to the extent to which expressed cancer-antigens can be adequatelyrecognised by the HLA of the individual, i.e. if there are epitopes incancer- or disease-associated antigens that are recognised by multiple,preferably at least three, HLA class I molecules of the subject.Therefore, in some cases, the methods of the disclosure may be used toidentify one or more or the subset of T cell clones that may bereactivated by a checkpoint inhibitor or to predict likely responders tocheckpoint inhibitor (immuno)therapies.

Accordingly in some cases the disclosure provides a method of predictingwhether a subject will respond to of cancer with a checkpoint inhibitor.In some cases the method comprises the step of identifying or selectingone or more polypeptides or polypeptide fragments that are associatedwith the disease or condition that is to be treated or that isassociated with achieving an immune or clinical response to treatmentwith a checkpoint inhibitor. In some cases the polypeptide is atumor-associated and/or mutational antigen. The polypeptide may bepresent in a sample obtained from the subject. The polypeptide may beone that is frequently (over-) expressed in a subject- and/ordisease-matched population. The polypeptide may consist of or comprise aPEPI (or PEPI3+) identified in a subject that is known to havepositively responded to a, or the, checkpoint inhibitor. The polypeptidemay comprise or consist of an amino acid sequence that is stored orrecorded in or retrieved from a database.

In some cases the method comprises determining whether thepolypeptide(s) comprise a sequence that is a T cell epitope capable ofbinding to multiple HLA class I molecules of the subject. In some casesthe presence of at least two, or at least three, or four or five or sixor seven or eight different such amino acid sequences is determined,and/or the presence of such an amino acid sequence in at least two, orat least three, or four or five different target polypeptide antigens.In some cases the method comprises determining whether thepolypeptide(s) comprise a sequence that is a T cell epitope capable ofbinding to at least two, or in some cases at least three or at leastfour HLA class II molecules of the subject. A response to treatment withthe or a checkpoint inhibitor may be predicted if the aboverequirement(s) is met. No response or no clinical response may bepredicted if the above requirement(s) is not met.

The disclosure also provides a method of identifying a fragment of apolypeptide or a T cell epitope in a polypeptide that may be targeted bythe subject's immune response following treatment with a checkpointinhibitor, or that will be targeted by T cells that are re-activated bytreatment with a checkpoint inhibitor.

The method may further comprise selecting, recommending and/oradministering a checkpoint inhibitor to a subject who is predicted torespond, or selecting, recommending and/or administering a differenttreatment to a subject that is predicted not to respond to a checkpointinhibitor. In other cases the disclosure provides a method of treatmentof a human subject in need thereof, the method comprising administeringto the subject a checkpoint inhibitor, wherein the subject has beenpredicted to respond to administration of a checkpoint inhibitor by themethod described herein.

Checkpoint inhibitors include, but are not limited to, PD-1 inhibitors,PD-L1 inhibitors, Lag-3 inhibitors, Tim-3 inhibitors, TIGIT inhibitors,BTLA inhibitors and CTLA-4 inhibitors, for example. Co-stimulatoryantibodies deliver positive signals through immune-regulatory receptorsincluding but not limited to ICOS, CD137, CD27 OX-40 and GITR. In oneembodiment the checkpoint inhibitor is a CTLA-4 inhibitor.

Design and Preparation of Pharmaceutical Compositions for an IndividualHuman Subject

In some aspects the disclosure provides a method of designing orpreparing a polypeptide, or a polynucleic acid that encodes apolypeptide, for inducing an immune response, a cytotoxic T cellresponse or a helper T cell response in a specific human subject. Thedisclosure also provides a human subject-specific drug, immunogeniccomposition, or pharmaceutical composition, kit or panel of peptides,methods of designing or preparing the same, compositions that may beobtained by those methods, and their use in a method of inducing animmune response, a cytotoxic T cell response, or a helper T cellresponse in the subject, or a method of treating, vaccinating orproviding immunotherapy to the subject. The pharmaceutical composition,kit or panel of peptides has as active ingredients one or morepolypeptides that together comprising two or more T cell epitopes(PEPIs) capable of binding to multiple HLA class I or multiple HLA classII molecules of the subject that are immunogenic for the subject asdescribed herein or that have been identified as immunogenic for thesubject by a method described herein.

The composition/kit may optionally further comprise at least onepharmaceutically acceptable diluent, carrier, or preservative and/oradditional polypeptides that do not comprise any PEPIs. The polypeptidesmay be engineered or non-naturally occurring. The kit may comprise oneor more separate containers each containing one or more of the activeingredient peptides. The composition/kit may be a personalised medicineto prevent, diagnose, alleviate, treat, or cure a disease of anindividual, such as a cancer.

Typically each PEPI is a fragment of a target polypeptide antigen andpolypeptides that comprise one or more of the PEPIs are the targetpolypeptide antigens for the treatment, vaccination or immunotherapy.The method may comprise the step of identifying one or more suitabletarget polypeptide antigens. Typically each target polypeptide antigenwill be associated with the same disease or condition, pathogenicorganism or group of pathogenic organisms or virus, or type of cancer.

The composition, kit or panel may comprise, or the method may compriseselecting, for each PEPI a sequence of up to 50, 45, 40, 35, 30, 25, 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or 9 consecutive amino acids ofthe target polypeptide antigen, such as a polypeptide described herein,which consecutive amino acids comprise the amino acid sequence of thePEPI.

In some cases the amino acid sequence is flanked at the N and/or Cterminus by additional amino acids that are not part of the consecutivesequence of the target polypeptide antigen. In some cases the sequenceis flanked by up to 41 or 35 or 30 or 25 or 20 or 15 or 10, or 9 or 8 or7 or 6 or 5 or 4 or 3 or 2 or 1 additional amino acid at the N and/or Cterminus or between target polypeptide fragments. In other cases eachpolypeptide either consists of a fragment of a target polypeptideantigen, or consists of two or more such fragments arranged end to end(arranged sequentially in the peptide end to end) or overlapping in asingle peptide (where two or more of the fragments comprise partiallyoverlapping sequences, for example where two PEPIs in the samepolypeptide are within 50 amino acids of each other).

When fragments of different polypeptides or from different regions ofthe same polypeptide are joined together in an engineered peptide thereis the potential for neoepitopes to be generated around the join orjunction. Such neoepitopes encompass at least one amino acid from eachfragment on either side of the join or junction, and may be referred toherein as junctional amino acid sequences. The neoepitopes may induceundesired T cell responses against healthy cells (autoimmunity). Thepeptides may be designed, or the peptides may be screened, to avoid oreliminate neoepitopes that correspond to a fragment of a proteinexpressed in normal healthy human cells and/or neoepitopes that arecapable of binding to at least two, or in some cases at least three, orat least four HLA class I molecules of the subject, or in some cases atleast two, or at least three or four or five HLA class II molecules ofthe subject. The methods of the disclosure may be used to identify orscreen for such neoepitopes as described herein. Alignment may bedetermined using known methods such as BLAST algorithms. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (ncbi.nlm.nih.gov/).

The at least two multiple HLA-binding PEPIs of the compositionpolypeptides may both target a single antigen (e.g a polypeptide vaccinecomprising two multiple HLA-binding PEPIs derived from a single antigen,for example a tumor associated antigen, targeted by thevaccine/immunotherapy) or may target different antigens (e.g. apolypeptide vaccine comprising one multiple HLA-binding PEPI derivedfrom one antigen, e.g. a tumor associated antigen, and a second multipleHLA-binding PEPI derived from a different antigen, e.g. a differenttumor associated antigen, both targeted by the vaccine/immunotherapy).

In some cases the active ingredient polypeptide(s) together comprise, orthe method comprises selecting, a total of or at least 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or moredifferent PEPIs. The PEPIs may be fragments of one or more differenttarget polypeptide antigens. By identifying the specific fragments ofeach target polypeptide antigen that are immunogenic for a specificsubject it is possible to incorporate multiple such fragments,optionally from multiple different target polypeptide antigens, in asingle active ingredient polypeptide or multiple active ingredientpolypeptides intended for use in combination or to maximise the numberof T cell clones that can be activated by one or more polypeptides of acertain length.

Currently most vaccines and immunotherapy compositions target only asingle polypeptide antigen. However according to the present disclosureit is in some cases beneficial to provide a pharmaceutical compositionor an active ingredient polypeptide that targets two or more differentpolypeptide antigens. For example, most cancers or tumors areheterogeneous, meaning that different cancer or tumor cells of a subject(over-)express different antigens. The tumour cells of different cancerpatients also express different combinations of tumour-associatedantigens. The anti-cancer immunogenic compositions that are most likelyto be effective are those that target multiple antigens expressed by thetumor, and therefore more cancer or tumor cells, in an individual humansubject or in a population.

The beneficial effect of combining multiple PEPIs in a single treatment(administration of one or more pharmaceutical compositions that togethercomprise multiple PEPIs), can be illustrated by the personalised vaccinepolypeptides described in Examples 17 and 18 below. Exemplary CTAexpression probabilities in ovarian cancer are as follows: BAGE: 30%;MAGE A9: 37%; MAGE A4: 34%; MAGE A10: 52%. If patient XYZ were treatedwith a vaccine comprising PEPIs in only BAGE and MAGE A9, then theprobability of having a mAGP (multiple expressed antigens with PEPI)would be 11%. If patent XYZ were treated with a vaccine comprising onlyPEPIs for the MAGE A4 and MAGE A10 CTAs, then the probability of havinga multiAGP would be 19%. However if a vaccine contained all 4 of theseCTAs (BAGE, MAGE A9, MAGE A4 and MAGE A10), then the probability ofhaving a mAGP would be 50%. In other words the effect would be greaterthan the combined probabilities of mAGP for both two-PEPI treatments(probability mAGP for BAGE/MAGE+probability mAGP for MAGE A4 and MAGEA10). Patient XYZ's PIT vaccine described in Example 17 contains afurther 9 PEPIs, and thus, the probability of having a mAGP is over99.95%.

Likewise exemplary CTA expression probabilities in breast cancer are asfollows: MAGE C2: 21%; MAGE A1: 37%; SPC1: 38%; MAGE A9: 44%. Treatmentof patient ABC with a vaccine comprising PEPIs in only MAGE C2: 21% andMAGE A1 has a mAGP probability of 7%. Treatment of patient ABC with avaccine comprising PEPIs in only SPC1: 38%; MAGE A9 has a mAGPprobability of 11%. Treatment of patient ABC with a vaccine comprisingPEPIs in MAGE C2: 21%; MAGE A1: 37%; SPC1: 38%; MAGE A9 has a mAGPprobability of 44% (44>7+11). Patient ABC's PIT vaccine described inExample 18 contains a further 8 PEPIs, and thus, the probability ofhaving a mAGP is over 99.93%.

Accordingly in some cases the PEPIs of the active ingredientpolypeptides are from two or more different target polypeptide antigens,for example different antigens associated with a specific disease orcondition, for example different cancer- or tumor-associated antigens orantigens expressed by a target pathogen. In some cases the PEPIs arefrom a total of or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39 or 40 or more different target polypeptideantigens. The different target polypeptide antigens may be any differentpolypeptides that it is useful to target or that can be selectivelytargeted with different PEPI3+s. In some cases different targetpolypeptide antigens are non-homologues or non-paralogues or have lessthan 95%, or 90%, or 85% or 80% or 75% or 70% or 60% or 50% sequenceidentity across the full length of each polypeptide. In some casesdifferent polypeptides are those that do not share any PEPI3+sAlternatively, in some cases the PEPI3+s are from different targetpolypeptide antigens when they are not shared with other polypeptideantigens targeted by the active ingredient polypeptides.

In some cases one or more or each of the immunogenic polypeptidefragments is from a polypeptide that is present in a sample taken fromthe specific human subject. This indicates that the polypeptide isexpressed in the subject, for example a cancer- or tumor-associatedantigen or a cancer testis antigen expressed by cancer cells of thesubject. In some cases one or more or each of the polypeptides is amutational neoantigen, or an expressional neoantigen of the subject. Oneor more or each fragment may comprise a neoantigen specific mutation.Since mutational neoantigens are subject specific, a composition thattargets one or more neoantigen specific mutations is personalised withregard to both their specific disease and their specific HLA set.

In other cases one or more or each of the immunogenic polypeptidefragments is from a target polypeptide antigen that is not generallyexpressed or is minimally expressed in normal healthy cells or tissue,but is expressed in a high proportion of (with a high frequency in)subjects or in the diseased cells of a subject having a particulardisease or condition, as described above. The method my compriseidentifying or selecting such a target polypeptide antigen. In somecases two or more or each of the immunogenic polypeptide fragments/PEPIsare from different cancer- or tumor-associated antigens that are each(over-)expressed with a high frequency in subjects having a type ofcancer or a cancer derived from a particular cell type or tissue. Insome cases the immunogenic polypeptide fragments are from a total of orat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39 or 40 different cancer- or tumor-associated polypeptides. Insome cases one or more or each or at least one, at least two, at leastthree, at least four, at least five or at least six or at least seven ofthe polypeptides are selected from the antigens listed in any one ofTables 2 to 7.

In some cases one or more or each of the target polypeptide antigens isa cancer testis antigen (CTA). In some cases the immunogenic polypeptidefragments/PEPIs are from at least 1, or at least 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 CTAs,or from a total of 3 or more different target polypeptide antigens,optionally wherein 1, 2, or all three or at least three are CTAs, orfrom 4 or more different polypeptide antigens, optionally wherein 1, 2,3 or all four or at least 1, 2, 3 or 4 are CTAs, or from 5 or moredifferent polypeptide antigens, optionally wherein 1, 2, 3, 4 or allfive or at least 1, 2, 3, 4, or 5 are CTAs, or from 6 or more differentpolypeptide antigens, optionally wherein 1, 2, 3, 4, 5 or all six or atleast 1, 2, 3, 4, 5, or 6 are CTAs, or from 7 or more differentpolypeptide antigens, optionally wherein 1, 2, 3, 4, 5, 6 or all 7 or atleast 1, 2, 3, 4, 5, 6 or 7 are CTAs, or from 8 or more differentpolypeptide antigens, optionally wherein 1, 2, 3, 4, 5, 6, 7 or all 8 orat least 1, 2, 3, 4, 5, 6, 7 or 8 are CTAs. In some cases one or more oreach of the target polypeptide antigens is expressed by a bacteria, avirus, or a parasite.

In some cases one or more of the polypeptide fragments comprises anamino acid sequence that is a T cell epitope capable of binding to atleast two, or at least three HLA class I of the subject and one or moreof the polypeptide fragments comprises an amino acid sequence that is aT cell epitope capable of binding to at least two, or at least three, orat least four HLA class II of the subject, wherein the HLA class I andHLA class II binding fragments may optionally overlap. A compositionprepared by such a method may elicit both a cytotoxic T cell responseand a helper T cell response in the specific human subject.

Immunogenic and Pharmaceutical Compositions, Methods of Treatment andModes of Administration

In some aspects the disclosure relates to a pharmaceutical composition,kit, or panels of polypeptides as described above having one or morepolypeptides as active ingredient(s). These may be for use in a methodof inducing an immune response, treating, vaccinating or providingimmunotherapy to a subject, and the pharmaceutical composition may be avaccine or immunotherapy composition. Such a treatment comprisesadministering one or more polypeptides or pharmaceutical compositionsthat together comprise all of the active ingredient polypeptides of thetreatment to the subject. Multiple polypeptides or pharmaceuticalcompositions may be administered together or sequentially, for exampleall of the pharmaceutical compositions or polypeptides may beadministered to the subject within a period of 1 year, or 6 months, or 3months, or 60 or 50 or 40 or 30 days.

The immunogenic or pharmaceutical compositions or kits described hereinmay comprise, in addition to one or more immunogenic peptides, apharmaceutically acceptable excipient, carrier, diluent, buffer,stabiliser, preservative, adjuvant or other materials well known tothose skilled in the art. Such materials are preferably non-toxic andpreferably do not interfere with the pharmaceutical activity of theactive ingredient(s). The pharmaceutical carrier or diluent may be, forexample, water containing solutions. The precise nature of the carrieror other material may depend on the route of administration, e.g. oral,intravenous, cutaneous or subcutaneous, nasal, intramuscular,intradermal, and intraperitoneal routes.

The pharmaceutical compositions of the disclosure may comprise one ormore “pharmaceutically acceptable carriers”. These are typically large,slowly metabolized macromolecules such as proteins, saccharides,polylactic acids, polyglycolic acids, polymeric amino acids, amino acidcopolymers, sucrose (Paoletti et al., 2001, Vaccine, 19:2118), trehalose(WO 00/56365), lactose and lipid aggregates (such as oil droplets orliposomes). Such carriers are well known to those of ordinary skill inthe art. The pharmaceutical compositions may also contain diluents, suchas water, saline, glycerol, etc. Additionally, auxiliary substances,such as wetting or emulsifying agents, pH buffering substances, and thelike, may be present. Sterile pyrogen-free, phosphate bufferedphysiologic saline is a typical carrier (Gennaro, 2000, Remington: TheScience and Practice of Pharmacy, 20th edition, ISBN:0683306472).

The pharmaceutical compositions of the disclosure may be lyophilized orin aqueous form, i.e. solutions or suspensions. Liquid formulations ofthis type allow the compositions to be administered direct from theirpackaged form, without the need for reconstitution in an aqueous medium,and are thus ideal for injection. The pharmaceutical compositions may bepresented in vials, or they may be presented in ready filled syringes.The syringes may be supplied with or without needles. A syringe willinclude a single dose, whereas a vial may include a single dose ormultiple doses.

Liquid formulations of the disclosure are also suitable forreconstituting other medicaments from a lyophilized form. Where apharmaceutical composition is to be used for such extemporaneousreconstitution, the disclosure provides a kit, which may comprise twovials, or may comprise one ready-filled syringe and one vial, with thecontents of the syringe being used to reconstitute the contents of thevial prior to injection.

The pharmaceutical compositions of the disclosure may include anantimicrobial, particularly when packaged in a multiple dose format.Antimicrobials may be used, such as 2-phenoxyethanol or parabens(methyl, ethyl, propyl parabens). Any preservative is preferably presentat low levels. Preservative may be added exogenously and/or may be acomponent of the bulk antigens which are mixed to form the composition(e.g. present as a preservative in pertussis antigens).

The pharmaceutical compositions of the disclosure may comprise detergente.g. Tween (polysorbate), DMSO (dimethyl sulfoxide), DMF(dimethylformamide). Detergents are generally present at low levels,e.g. <0.01%, but may also be used at higher levels, e.g. 0.01-50%.

The pharmaceutical compositions of the disclosure may include sodiumsalts (e.g. sodium chloride) and free phosphate ions in solution (e.g.by the use of a phosphate buffer).

In certain embodiments, the pharmaceutical composition may beencapsulated in a suitable vehicle either to deliver the peptides intoantigen presenting cells or to increase the stability. As will beappreciated by a skilled artisan, a variety of vehicles are suitable fordelivering a pharmaceutical composition of the disclosure. Non-limitingexamples of suitable structured fluid delivery systems may includenanoparticles, liposomes, microemulsions, micelles, dendrimers and otherphospholipid-containing systems. Methods of incorporating pharmaceuticalcompositions into delivery vehicles are known in the art.

In order to increase the immunogenicity of the composition, thepharmacological compositions may comprise one or more adjuvants and/orcytokines.

Suitable adjuvants include an aluminum salt such as aluminum hydroxideor aluminum phosphate, but may also be a salt of calcium, iron or zinc,or may be an insoluble suspension of acylated tyrosine, or acylatedsugars, or may be cationically or anionically derivatised saccharides,polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A(MPL), lipid A derivatives (e.g. of reduced toxicity), 3-O-deacylatedMPL [3D-MPL], quil A, Saponin, QS21, Freund's Incomplete Adjuvant (DifcoLaboratories, Detroit, Mich.), Merck Adjuvant 65 (Merck and Company,Inc., Rahway, N.J.), AS-2 (Smith-Kline Beecham, Philadelphia, Pa.), CpGoligonucleotides, bioadhesives and mucoadhesives, microparticles,liposomes, polyoxyethylene ether formulations, polyoxyethylene esterformulations, muramyl peptides or imidazoquinolone compounds (e.g.imiquamod and its homologues). Human immunomodulators suitable for useas adjuvants in the disclosure include cytokines such as interleukins(e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc), macrophage colonystimulating factor (M-CSF), tumour necrosis factor (TNF), granulocyte,macrophage colony stimulating factor (GM-CSF) may also be used asadjuvants.

In some embodiments, the compositions comprise an adjuvant selected fromthe group consisting of Montanide ISA-51 (Seppic, Inc., Fairfield, N.J.,United States of America), QS-21 (Aquila Biopharmaceuticals, Inc.,Lexington, Mass., United States of America), GM-CSF, cyclophosamide,bacillus Calmette-Guerin (BCG), corynbacterium parvum, levamisole,azimezone, isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpethemocyanins (KLH), Freunds adjuvant (complete and incomplete), mineralgels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, dinitrophenol, diphtheria toxin(DT).

By way of example, the cytokine may be selected from the groupconsisting of a transforming growth factor (TGF) such as but not limitedto TGF-α and TGF-0; insulin-like growth factor-I and/or insulin-likegrowth factor-II; erythropoietin (EPO); an osteoinductive factor; aninterferon such as but not limited to interferon-α, -β, and -γ; a colonystimulating factor (CSF) such as but not limited to macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF). In some embodiments, the cytokine is selected from the groupconsisting of nerve growth factors such as NGF-β; platelet-growthfactor; a transforming growth factor (TGF) such as but not limited toTGF-α. and TGF-β; insulin-like growth factor-I and insulin-like growthfactor-II; erythropoietin (EPO); an osteoinductive factor; an interferon(IFN) such as but not limited to IFN-α, IFN-β, and IFN-γ; a colonystimulating factor (CSF) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); aninterleukin (Il) such as but not limited to IL-1, IL-1.alpha., IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18; LIF; kit-ligand or FLT-3;angiostatin; thrombospondin; endostatin; a tumor necrosis factor (TNF);and LT.

It is expected that an adjuvant or cytokine can be added in an amount ofabout 0.01 mg to about 10 mg per dose, preferably in an amount of about0.2 mg to about 5 mg per dose. Alternatively, the adjuvant or cytokinemay be at a concentration of about 0.01 to 50%, preferably at aconcentration of about 2% to 30%.

In certain aspects, the pharmaceutical compositions of the disclosureare prepared by physically mixing the adjuvant and/or cytokine with thePEPIs under appropriate sterile conditions in accordance with knowntechniques to produce the final product.

Examples of suitable compositions of polypeptide fragments and methodsof administration are provided in Esseku and Adeyeye (2011) and Van denMooter G. (2006). Vaccine and immunotherapy composition preparation isgenerally described in Vaccine Design (“The subunit and adjuvantapproach” (eds Powell M. F. & Newman M. J. (1995) Plenum Press NewYork). Encapsulation within liposomes, which is also envisaged, isdescribed by Fullerton, U.S. Pat. No. 4,235,877.

In some embodiments, the compositions disclosed herein are prepared as anucleic acid vaccine. In some embodiments, the nucleic acid vaccine is aDNA vaccine. In some embodiments, DNA vaccines, or gene vaccines,comprise a plasmid with a promoter and appropriate transcription andtranslation control elements and a nucleic acid sequence encoding one ormore polypeptides of the disclosure. In some embodiments, the plasmidsalso include sequences to enhance, for example, expression levels,intracellular targeting, or proteasomal processing. In some embodiments,DNA vaccines comprise a viral vector containing a nucleic acid sequenceencoding one or more polypeptides of the disclosure. In additionalaspects, the compositions disclosed herein comprise one or more nucleicacids encoding peptides determined to have immunoreactivity with abiological sample. For example, in some embodiments, the compositionscomprise one or more nucleotide sequences encoding 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more peptidescomprising a fragment that is a T cell epitope capable of binding to atleast three HLA class I molecules and/or at least three HLA class IImolecules of a patient. In some embodiments, the peptides are derivedfrom an antigen that is expressed in cancer. In some embodiments the DNAor gene vaccine also encodes immunomodulatory molecules to manipulatethe resulting immune responses, such as enhancing the potency of thevaccine, stimulating the immune system or reducing immunosuppression.Strategies for enhancing the immunogenicity of of DNA or gene vaccinesinclude encoding of xenogeneic versions of antigens, fusion of antigensto molecules that activate T cells or trigger associative recognition,priming with DNA vectors followed by boosting with viral vector, andutilization of immunomodulatory molecules. In some embodiments, the DNAvaccine is introduced by a needle, a gene gun, an aerosol injector, withpatches, via microneedles, by abrasion, among other forms. In some formsthe DNA vaccine is incorporated into liposomes or other forms ofnanobodies. In some embodiments, the DNA vaccine includes a deliverysystem selected from the group consisting of a transfection agent;protamine; a protamine liposome; a polysaccharide particle; a cationicnanoemulsion; a cationic polymer; a cationic polymer liposome; acationic nanoparticle; a cationic lipid and cholesterol nanoparticle; acationic lipid, cholesterol, and PEG nanoparticle; a dendrimernanoparticle. In some embodiments, the DNA vaccines is administered byinhalation or ingestion. In some embodiments, the DNA vaccine isintroduced into the blood, the thymus, the pancreas, the skin, themuscle, a tumor, or other sites.

In some embodiments, the compositions disclosed herein are prepared asan RNA vaccine. In some embodiments, the RNA is non-replicating mRNA orvirally derived, self-amplifying RNA. In some embodiments, thenon-replicating mRNA encodes the peptides disclosed herein and contains5′ and 3′ untranslated regions (UTRs). In some embodiments, the virallyderived, self-amplifying RNA encodes not only the peptides disclosedherein but also the viral replication machinery that enablesintracellular RNA amplification and abundant protein expression. In someembodiments, the RNA is directly introduced into the individual. In someembodiments, the RNA is chemically synthesized or transcribed in vitro.In some embodiments, the mRNA is produced from a linear DNA templateusing a T7, a T3, or an Sp6 phage RNA polymerase, and the resultingproduct contains an open reading frame that encodes the peptidesdisclosed herein, flanking UTRs, a 5′ cap, and a poly(A) tail. In someembodiments, various versions of 5′ caps are added during or after thetranscription reaction using a vaccinia virus capping enzyme or byincorporating synthetic cap or anti-reverse cap analogues. In someembodiments, an optimal length of the poly(A) tail is added to mRNAeither directly from the encoding DNA template or by using poly(A)polymerase. The RNA encodes one or more peptides comprising a fragmentthat is a T cell epitope capable of binding to at least three HLA classI and/or at least three HLA class II molecules of a patient. In someembodiments, the fragments are derived from an antigen that is expressedin cancer. In some embodiments, the RNA includes signals to enhancestability and translation. In some embodiments, the RNA also includesunnatural nucleotides to increase the half-life or modified nucleosidesto change the immunostimulatory profile. In some embodiments, the RNAsis introduced by a needle, a gene gun, an aerosol injector, withpatches, via microneedles, by abrasion, among other forms. In some formsthe RNA vaccine is incorporated into liposomes or other forms ofnanobodies that facilitate cellular uptake of RNA and protect it fromdegradation. In some embodiments, the RNA vaccine includes a deliverysystem selected from the group consisting of a transfection agent;protamine; a protamine liposome; a polysaccharide particle; a cationicnanoemulsion; a cationic polymer; a cationic polymer liposome; acationic nanoparticle; a cationic lipid and cholesterol nanoparticle; acationic lipid, cholesterol, and PEG nanoparticle; a dendrimernanoparticle; and/or naked mRNA; naked mRNA with in vivoelectroporation; protamine-complexed mRNA; mRNA associated with apositively charged oil-in-water cationic nanoemulsion; rnRNA associatedwith a chemically modified dendrirmer and complexed with polyethyleneglycol (PEG)-lipid; protamine-complexed mRNA in a PEG-lipidnanoparticle; mRNA associated with a cationic polymer such aspolyethylenimine (PEI); rnRNA associated with a cationic polymer such asPEI and a lipid component; mRNA associated with a polysaccharide (forexample, chitosan) particle or gel; mRNA in a cationic lipidnanoparticle (for example, 1,2-dioleoyloxy-3-trimethylamnoniunpropane(DOTAP) or dioleoylphosphatidylethanolamine (DOPE) lipids); mRNAcomplexed with cationic lipids and cholesterol; or mRNA complexed withcationic lipids, cholesterol and PEG-lipid. In some embodiments, the RNAvaccine is administered by inhalation or ingestion. In some embodiments,the RNA is introduced into the blood, the thymus, the pancreas, theskin, the muscle, a tumor, or other sites, and/or by an intradermal,intramuscular, subcutaneous, intranasal, intranodal, intravenous,intrasplenic, intratumoral or other delivery route.

Polynucleotide or oligonucleotide components may be naked nucleotidesequences, or be in combination with cationic lipids, polymers ortargeting systems. They may be delivered by any available technique. Forexample, the polynucleotide or oligonucleotide may be introduced byneedle injection, preferably intradermally, subcutaneously orintramuscularly. Alternatively, the polynucleotide or oligonucleotidemay be delivered directly across the skin using a delivery device suchas particle-mediated gene delivery. The polynucleotide oroligonucleotide may be administered topically to the skin, or to mucosalsurfaces for example by intranasal, oral, or intrarectal administration.

Uptake of polynucleotide or oligonucleotide constructs may be enhancedby several known transfection techniques, for example those includingthe use of transfection agents. Examples of these agents includecationic agents, for example, calcium phosphate and DEAE-Dextran andlipofectants, for example, lipofectam and transfectam. The dosage of thepolynucleotide or oligonucleotide to be administered can be altered.

Administration is typically in a “prophylactically effective amount” ora “therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to resultin a clinical response or to show clinical benefit to the individual,e.g. an effective amount to prevent or delay onset of the disease orcondition, to ameliorate one or more symptoms, to induce or prolongremission, or to delay relapse or recurrence.

The dose may be determined according to various parameters, especiallyaccording to the substance used; the age, weight and condition of theindividual to be treated; the route of administration; and the requiredregimen. The amount of antigen in each dose is selected as an amountwhich induces an immune response. A physician will be able to determinethe required route of administration and dosage for any particularindividual. The dose may be provided as a single dose or may be providedas multiple doses, for example taken at regular intervals, for example2, 3 or 4 doses administered hourly. Typically peptides, polynucleotidesor oligonucleotides are typically administered in the range of 1 pg to 1mg, more typically 1 pg to 10 μg for particle mediated delivery and 1 μgto 1 mg, more typically 1-100 μg, more typically 5-50 μg for otherroutes. Generally, it is expected that each dose will comprise 0.01-3 mgof antigen. An optimal amount for a particular vaccine can beascertained by studies involving observation of immune responses insubjects.

Examples of the techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 20th Edition, 2000, pub.Lippincott, Williams & Wilkins.

In some cases in accordance with the disclosure, more than one peptideor composition of peptides is administered. Two or more pharmaceuticalcompositions may be administered together/simultaneously and/or atdifferent times or sequentially. Thus, the disclosure includes sets ofpharmaceutical compositions and uses thereof. The use of combination ofdifferent peptides, optionally targeting different antigens, isimportant to overcome the challenges of genetic heterogeneity of tumorsand HLA heterogeneity of individuals. Multiple pharmaceuticalcompositions of PEPIs, manufactured for use in one regimen, may define adrug product.

Routes of administration include but are not limited to intranasal,oral, subcutaneous, intradermal, and intramuscular. The subcutaneousadministration is particularly preferred. Subcutaneous administrationmay for example be by injection into the abdomen, lateral and anterioraspects of upper arm or thigh, scapular area of back, or upperventrodorsal gluteal area.

The skilled artisan will recognize that compositions of the disclosuremay also be administered in one, or more doses, as well as, by otherroutes of administration. For example, such other routes include,intracutaneously, intravenously, intravascularly, intraarterially,intraperitnoeally, intrathecally, intratracheally, intracardially,intralobally, intramedullarly, intrapulmonarily, and intravaginally.Depending on the desired duration of the treatment, the compositionsaccording to the disclosure may be administered once or several times,also intermittently, for instance on a monthly basis for several monthsor years and in different dosages.

Solid dosage forms for oral administration include capsules, tablets,caplets, pills, powders, pellets, and granules. In such solid dosageforms, the active ingredient is ordinarily combined with one or morepharmaceutically acceptable excipients, examples of which are detailedabove. Oral preparations may also be administered as aqueoussuspensions, elixirs, or syrups. For these, the active ingredient may becombined with various sweetening or flavoring agents, coloring agents,and, if so desired, emulsifying and/or suspending agents, as well asdiluents such as water, ethanol, glycerin, and combinations thereof.

One or more compositions of the disclosure may be administered, or themethods and uses for treatment according to the disclosure may beperformed, alone or in combination with other pharmacologicalcompositions or treatments, for example chemotherapy and/orimmunotherapy and/or vaccine. The other therapeutic compositions ortreatments may for example be one or more of those discussed herein, andmay be administered either simultaneously or sequentially with (beforeor after) the composition or treatment of the disclosure.

In some cases the treatment may be administered in combination withcheckpoint blockade therapy/checkpopint inhibitors, co-stimulatoryantibodies, cytotoxic or non-cytotoxic chemotherapy and/or radiotherapy,targeted therapy or monoclonal antibody therapy. It has beendemonstrated that chemotherapy sensitizes tumors to be killed by tumorspecific cytotoxic T cells induced by vaccination (Ramakrishnan et al. JClin Invest. 2010; 120(4):1111-1124). Examples of chemotherapy agentsinclude alkylating agents including nitrogen mustards such asmechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan(L-sarcolysin) and chlorambucil; anthracyclines; epothilones;nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine(methyl-CCNU) and streptozocin (streptozotocin); triazenes such asdecarbazine (DTIC; dimethyltriazenoimidazole-carboxamide;ethylenimines/methylmelamines such as hexamethylmelamine, thiotepa;alkyl sulfonates such as busulfan; Antimetabolites including folic acidanalogues such as methotrexate (amethopterin); alkylating agents,antimetabolites, pyrimidine analogs such as fluorouracil(5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) andcytarabine (cytosine arabinoside); purine analogues and relatedinhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine(6-thioguanine; TG) and pentostatin (2′-deoxycoformycin);epipodophylotoxins; enzymes such as L-asparaginase; biological responsemodifiers such as IFNα, IL-2, G-CSF and GM-CSF; platinum coordinationcomplexes such as cisplatin (cis-DDP), oxaliplatin and carboplatin;anthracenediones such as mitoxantrone and anthracycline; substitutedurea such as hydroxyurea; methylhydrazine derivatives includingprocarbazine (N-methylhydrazine, MIH) and procarbazine; adrenocorticalsuppressants such as mitotane (o,p′-DDD) and aminoglutethimide; taxoland analogues/derivatives; hormones/hormonal therapy andagonists/antagonists including adrenocorticosteroid antagonists such asprednisone and equivalents, dexamethasone and aminoglutethimide,progestin such as hydroxyprogesterone caproate, medroxyprogesteroneacetate and megestrol acetate, estrogen such as diethylstilbestrol andethinyl estradiol equivalents, antiestrogen such as tamoxifen, androgensincluding testosterone propionate and fluoxymesterone/equivalents,antiandrogens such as flutamide, gonadotropin-releasing hormone analogsand leuprolide and non-steroidal antiandrogens such as flutamide;natural products including vinca alkaloids such as vinblastine (VLB) andvincristine, epipodophyllotoxins such as etoposide and teniposide,antibiotics such as dactinomycin (actinomycin D), daunorubicin(daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin(mithramycin) and mitomycin (mitomycin C), enzymes such asL-asparaginase, and biological response modifiers such as interferonalphenomes.

In some cases the method of treatment is a method of vaccination or amethod of providing immunotherapy. As used herein, “immunotherapy” isthe treatment of a disease or condition by inducing or enhancing animmune response in an individual. In certain embodiments, immunotherapyrefers to a therapy that comprises the administration of one or moredrugs to an individual to elicit T cell responses. In a specificembodiment, immunotherapy refers to a therapy that comprises theadministration or expression of polypeptides that contain one or morePEPIs to an individual to elicit a T cell response to recognize and killcells that display the one or more PEPIs on their cell surface inconjunction with a class I HLA. In another specific embodiment,immunotherapy comprises the administration of one or more PEPIs to anindividual to elicit a cytotoxic T cell response against cells thatdisplay tumor associated antigens (TAAs) or cancer testis antigens(CTAs) comprising the one or more PEPIs on their cell surface. Inanother embodiment, immunotherapy refers to a therapy that comprises theadministration or expression of polypeptides that contain one or morePEPIs presented by class II HLAs to an individual to elicit a T helperresponse to provide co-stimulation to cytotoxic T cells that recognizeand kill diseased cells that display the one or more PEPIs on their cellsurface in conjunction with a class I HLAs. In still another specificembodiment, immunotherapy refers to a therapy that comprisesadministration of one or more drugs to an individual that re-activateexisting T cells to kill target cells. The theory is that the cytotoxicT cell response will eliminate the cells displaying the one or morePEPIs, thereby improving the clinical condition of the individual. Insome instances, immunotherapy may be used to treat tumors. In otherinstances, immunotherapy may be used to treat intracellularpathogen-based diseases or disorders.

In some cases the disclosure relates to the treatment of cancer or thetreatment of solid tumors. The treatment may be of cancers or malignantor benign tumors of any cell, tissue, or organ type. The cancer may ormay not be metastatic. Exemplary cancers include carcinomas, sarcomas,lymphomas, leukemias, germ cell tumors, or blastomas. The cancer may ormay not be a hormone related or dependent cancer (e.g., an estrogen orandrogen related cancer).

In other cases the disclosure relates to the treatment of a viral,bacterial, fungal or parasitic infection, or any other disease orcondition that may be treated by immunotherapy.

Systems

The disclosure provides a system comprising a storage module configuredto store data comprising the class I and/or class II HLA genotype of asubject and the amino acid sequence of one or more test polypeptides;and a computation module configured to identify and/or quantify aminoacid sequences in the one or more test polypeptides that are capable ofbinding to multiple HLA of the subject. The system may be for obtainingdata from at least one sample from at least one subject. The system maycomprise a an HLA genotyping module for determining the class I and/orclass II HLA genotype of a subject. The storage module may be configuredto store the data output from the genotyping module. The HLA genotypingmodule may receive a biological sample obtained from the subject anddetermines the subject's class I and/or class II HLA genotype. Thesample typically contains subject DNA. The sample may be, for example, ablood, serum, plasma, saliva, urine, expiration, cell or tissue sample.The system may further comprise an output module configured to displaythe sequence of one or more fragments of the one or more polypeptidesthat are predicted to be immunogenic for the subject, or any outputprediction or treatment selection or recommendation described herein orthe value of any pharmodynamic biomarker described herein.

Further Embodiments of the Disclosure

1. A method of predicting whether a polypeptide or a fragment of apolypeptide is immunogenic for a specific human subject, the methodcomprising the steps of

-   -   (i) determining whether the polypeptide comprises:        -   (a) an amino acid sequence that is a T cell epitope capable            of binding to at least two HLA class I molecules of the            subject; or        -   (b) an amino acid sequence that is a T cell epitope capable            of binding to at least two HLA class II molecules of the            subject; and    -   (ii) predicting        -   A. that the polypeptide is immunogenic for the subject if            the polypeptide comprises at least one sequence that meets            the requirements of step (i); or        -   B. that the polypeptide is not immunogenic for the subject            if the polypeptide does not comprise at least one sequence            that meets the requirements of step (i)

2. A method of identifying a fragment of a polypeptide as immunogenicfor a specific human subject, the method comprising the steps of

-   -   (i) determining that the polypeptide comprises:        -   (a) an amino acid sequence that is a T cell epitope capable            of binding to at least two HLA class I molecules of the            subject; or        -   (b) an amino acid sequence that is a T cell epitope capable            of binding to at least two HLA class II molecules of the            subject; and    -   (ii) identifying said sequence as a fragment of the polypeptide        that is immunogenic for the subject.

3. The method of item 1 or item 2, wherein the T cell epitope is capableof binding to at least two HLA class I molecules of the subject andconsists of 9 consecutive amino acids of the polypeptide, or wherein theT cell epitope is capable of binding to at least two HLA class IImolecules of the subject and consists of 15 consecutive amino acids ofthe polypeptide.

4. The method of any one of the preceding items, wherein step (i)comprises determining that the polypeptide comprises an amino acidsequence that is a T cell epitope capable of binding to at least two HLAclass I molecules of the subject.

5. The method of any one of the preceding items, wherein step (i)comprises determining that the polypeptide comprises an amino acidsequence that is a T cell epitope capable of binding to at least threeHLA class I molecules of the subject.

6. The method of any one of items 1 to 3, wherein step (i) comprisesdetermining that the polypeptide comprises an amino acid sequence thatis a T cell epitope capable of binding to at least three HLA class IImolecules of the subject.

7. The method of item 4 or item 5 further comprising identifying afragment of the polypeptide that is a T cell epitope capable of bindingto at least one HLA class II molecule of the subject, wherein the HLAclass II-binding epitope comprises the amino acid sequence of the HLAclass I-binding T cell epitope.

8. The method of any one of the preceding items, wherein the polypeptideis expressed by a pathogenic organism, a virus or a cancer cell, isassociated with an autoimmune disorder, or is an allergen or aningredient of a pharmaceutical composition.

9. The method of any one of the preceding items, wherein the polypeptideis selected from the antigens listed in Tables 2 to 6.

10. The method of any one of the preceding items, wherein thepolypeptide is an antigen or neoantigen expressed by a cancer cell,optionally wherein the cancer cell, the antigen or the neoantigen is ina sample taken from the subject.

11. The method of any one of the preceding items, wherein thepolypeptide is a mutational neoantigen, optionally wherein

-   -   (a) the neoantigen is present in a sample obtained from the        subject; and/or    -   (b) the immunogenic fragment comprises a neoantigen specific        mutation.

12. The method of any one of items 1 to 11, wherein all of the fragmentsof the polypeptide that are a T cell epitope capable of binding to atleast two HLA class I molecules and/or all of the fragments of thepolypeptide that are a T cell epitope capable of binding to at least twoHLA class II molecules of the subject are identified, optionally whereinthe method is repeated for each polypeptide that is an active ingredientof a specific pharmaceutical composition.

13. The method of any one of the preceding items, further comprisingpredicting whether the subject will have a cytotoxic T cell response ora helper T cell response to administration of one or more polypeptide ora pharmaceutical composition or kit comprising one or more polypeptidesas active ingredients, wherein

-   -   A. a cytotoxic T cell response is predicted if the        polypeptide(s) comprises at least one amino acid sequence that        is a T cell epitope capable of binding to at least three HLA        class I molecules of the subject;    -   B. a helper T cell response is predicted if the polypeptide(s)        comprises at least one amino acid sequence that is a T cell        epitope capable of binding to at least three HLA class II        molecules of the subject;    -   C. no cytotoxic T cell response is predicted if the        polypeptide(s) does not comprise any amino acid sequence that is        a T cell epitope capable of binding to at least three HLA class        I molecules of the subject; or    -   D. no helper T cell response is predicted if the polypeptide(s)        does not comprise any amino acid sequence that is a T cell        epitope capable of binding to at least three HLA class II        molecules of the subject.

14. The method of item 13, wherein the subject is predicted to have acytotoxic T cell and/or a helper T cell response, and the method furthercomprises determine the likelihood that the subject will have acytotoxic T cell response and/or a helper T cell response that targets apolypeptide antigen that is expressed in the subject, the methodcomprising

-   -   (i) identifying one or more polypeptide antigens that comprises        an amino acid sequence that is        -   (a) a T cell epitope capable of binding to at least three            HLA class I or at least three HLA class II molecules of the            subject; and        -   (b) comprised in the amino acid sequence of the            polypeptide(s)    -   (ii) using population expression frequency data for the one or        more polypeptide antigens identified in step (i) to determine        the likelihood that the subject will have a cytotoxic T cell        response and/or a helper T cell response that targets a        polypeptide antigen that is expressed in the subject.

15. The method of item 13 wherein the polypeptide is a component of apharmaceutical composition and the method comprises determining thelikelihood that the subject will develop anti-drug antibodies (ADA)following administration of the polypeptide, wherein a predicted Thelper cell response corresponds to a higher likihood of ADA and nopredicted T helper cell response corresponds to a lower likelihood ofADA.

16. The method of item 15, wherein the polypeptide is a checkpointinhibitor.

17. The method of any one of items 1 to 14 further comprising predictingwhether the subject will have a clinical response to administration of apharmaceutical composition, kit or panel of polypeptides comprising oneor more polypeptides as active ingredients, the method comprisingdetermining whether the one or more active ingredient polypeptidestogether comprise at least two different amino acid sequences each ofwhich is a T cell epitope capable of binding to at least three HLA classI molecules of the subject; and predicting

-   -   A. that the subject will have a clinical response to        administration of the pharmaceutical composition, kit or panel        of polypeptides if the one or more active ingredient        polypeptides together comprise at least two different sequences        each of which is a T cell epitope capable of binding to at least        three HLA class I molecules of the subject; or    -   B. that the subject will not have a clinical response to        administration of the pharmaceutical composition, kit or panel        of polypeptides if the one or more active ingredient        polypeptides together comprise no more than one sequence that is        a T cell epitope capable of binding to at least three HLA class        I molecules of the subject.

18. The method of item 17, wherein the at least two different amino acidsequences are comprised in the amino acid sequence of two differentpolypeptide antigens targeted by the active ingredient polypeptide(s).

19. The method of any one of items 1 to 14, 17 and 18, furthercomprising determining the likelihood that the specific human subjectwill have a clinical response to administration of a pharmaceuticalcomposition, kit or panel of polypeptides comprising one or morepolypeptides as active ingredients, wherein one or more of the followingfactors corresponds to a higher likelihood of a clinical response:

-   -   (a) presence in the active ingredient polypeptide(s) of a higher        number of amino acid sequences and/or different amino acid        sequences that are each a T cell epitope capable of binding to        at least three HLA class I of the subject;    -   (b) a higher number of target polypeptide antigens, comprising        at least one amino acid sequence that is both        -   A. comprised in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class I of the subject; optionally wherein the target            polypeptide antigens are expressed in the subject, further            optionally wherein the target polypeptides antigens are in            one or more samples obtained from the subject;    -   (c) a higher probability that the subject expresses target        polypeptide antigens, optionally a threshold number of the        target polypeptide antigens and/or optionally target polypeptide        antigens that have been determined to comprise at least one        amino acid sequence that is both        -   A. comprised in in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class I of the subject; and/or    -   (d) a higher number of target polypeptide antigens that the        subject is predicted to express, optionally a higher number of        target polypeptide antigens that the subject expresses with a        threshold probability, and/or optionally the target polypeptide        antigens that have been determined to comprise at least one        amino acid sequence that is both        -   A. comprised in in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class I of the subject.

20. The method of any one of items 1 to 14, and 17 to 19, comprisingdetermining the likelihood that the specific human subject will have aclinical response to administration of a pharmaceutical composition, kitor panel of polypeptides comprising one or more polypeptides as activeingredients, wherein the method comprises

-   -   (i) identifying which polypeptide antigens targeted by the        active ingredient polypeptide(s) comprise an amino acid sequence        that is both        -   A. comprised in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class I of the subject;    -   (ii) using population expression data for each antigen        identified in step (i) to determine the probability that the        subject expresses one or more of the antigens identified in        step (i) that together comprise at least two different amino        acid sequences of step (i); and    -   (iii) determining the likelihood that the subject will have a        clinical response to administration of the pharmaceutical        composition, kit or panel of polypeptides, wherein a higher        probability determined in step (ii) corresponds to a more likely        clinical response.

21. The method of item 20, wherein step (ii) comprises using populationexpression data for each antigen identified in step (i) to determine theprobability that the subject expresses two or more of the antigensidentified in step (i) that together comprise at least two differentamino acid sequences of step (i).

22. The method of item 21, wherein the at least two different amino acidsequences are comprised in the amino acid sequence of two differentpolypeptide antigens targeted by the active ingredient polypeptide(s).

23. The method of any one of items 19 to 22, wherein one or more of thefollowing factors

further correspond to a higher likelihood of a clinical response:

-   -   (a) presence in the active ingredient polypeptide(s) of a higher        number of amino acid sequences and/or different amino acid        sequences that are each a T cell epitope capable of binding to        at least three HLA class II of the subject;    -   (b) a higher number of target polypeptide antigens comprising at        least one amino acid sequence that is both        -   A. comprised in an active ingredient polypeptide; and        -   B. a T cell epitope-capable of binding to at least three HLA            class II of the subject, optionally wherein the target            polypeptide antigens are expressed in the subject,            optionally wherein the target polypeptides antigens are in            one or more samples obtained from the subject;    -   (c) a higher number of target polypeptide antigens comprising        -   i. at least one amino acid sequence that is both            -   A. comprised in an active ingredient polypeptide; and            -   B. a T cell epitope capable of binding to at least three                HLA class I of the subject; and        -   ii. at least one amino acid sequence that is both            -   A. comprised in an active ingredient polypeptide; and            -   B. a T cell epitope capable of binding to at least three                HLA class II of the subject;    -   (d) a higher probability that the subject expresses target        polypeptide antigens, optionally a threshold number of the        target polypeptide antigens, that have been determined to        comprise at least one amino acid sequence that is both        -   A. comprised in in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class II of the subject    -   (e) a higher probability that the subject expresses target        polypeptide antigens, optionally a threshold number of the        target polypeptide antigens, that have been determined to        comprise        -   i. at least one amino acid sequence that is both            -   A. comprised in an active ingredient polypeptide; and            -   B. a T cell epitope capable of binding to at least three                HLA class I of the subject; and        -   ii. at least one amino acid sequence that is both        -   A. comprised in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class II of the subject;    -   (f) a higher number of target polypeptide antigens that the        subject is predicted to express, optionally a higher number of        target polypeptide antigens that the subject expresses with a        threshold probability, and that have been determined to comprise        at least one amino acid sequence that is both        -   A. comprised in an active ingredient polypeptide; and        -   B. a T cell epitope capable of binding to at least three HLA            class II of the subject; and/or    -   (g) a higher number of target polypeptide antigens that the        subject is predicted to express, optionally a higher number of        target polypeptide antigens that the subject expresses with a        threshold probability, and that have been determined to comprise        -   i. at least one amino acid sequence that is both            -   A. comprised in an active ingredient polypeptide; and            -   B. a T cell epitope capable of binding to at least three                HLA class I of the subject; and        -   ii. at least one amino acid sequence that is both            -   A. comprised in an active ingredient polypeptide; and            -   B. a T cell epitope capable of binding to at least three                HLA class II of the subject.

24. The method of any one of items 19 to 23, further comprisingrepeating the method for one or more further pharmaceuticalcompositions, kits or panels of polypeptides and ranking thecompositions, kits or panels of polypeptides by their likelihood toinduce a clinical response in the subject.

25. The method of any one of items 1 to 24, further comprisingpredicting whether administration of the polypeptide, pharmaceuticalcomposition, kit or panel of polypeptides will induce a toxic immuneresponse in the subject, wherein

-   -   (a) the polypeptide(s) comprises at least one amino acid        sequence that        -   i. is capable of binding to at least three HLA class I of            the subject; and        -   ii. corresponds to a fragment of a human polypeptide            expressed in healthy cells; and a toxic immune response is            predicted; or    -   (b) the polypeptide(s) do not comprise any amino acid sequence        that        -   A. is capable of binding to at least three HLA class I of            the subject; and        -   B. corresponds to a fragment of a human polypeptide            expressed in healthy cells; and no toxic immune response is            predicted.

26. The method of any one of the preceding items further comprisingselecting or recommending for treatment of the specific human subjectadministration to the subject of a polypeptide that comprises apolypeptide fragment that is identified as immunogenic for the subject,or of a polypeptide that is predicted to be immunogenic, or to induce acytotoxic T cell or helper T cell response, or of a pharmaceuticalcomposition, kit or panel of polypeptides that is predicted to induce aclinical response, or of a polypeptide or pharmaceutical compositionthat is predicted not to induce a toxic immune response or not to induceADA in the subject.

27. The method of item 26, further comprising administering one or moreof the selected or recommended polypeptides or pharmaceuticalcompositions or the polypeptides of one or more kits or panels ofpolypeptides to the subject.

28. A method of treatment of a human subject in need thereof, the methodcomprising administering to the subject a polypeptide that comprises apolypeptide fragment that has been identified as immunogenic, or apolypeptide that has been predicted to be immunogenic, or a polypeptideor pharmaceutical composition that has been predicted to induce acytotoxic T cell or helper T cell response, or a pharmaceuticalcomposition, kit or panel of polypeptides that has been predicted toinduce a clinical response, or a pharmaceutical composition, kit orpanel of polypeptides that has been determined to have a thresholdminimum likelihood of inducing a clinical response, or a polypeptide orpharmaceutical composition that is predicted not to induce a toxicimmune response or ADA development in the subject using a methodaccording to any one of items 1 to 23, or one or more polypeptides orpharmaceutical compositions that have been selected or recommended fortreatment of the subject using a method according to item 26.

29. The method of any one of items 1 to 11, wherein the polypeptide isassociated with or suspected of being associated with an autoimmunedisorder or an autoimmune response in the subject and determining thatthe polypeptide comprises an amino acid sequence that is a T cellepitope capable of binding to at least three HLA class I molecules ofthe subject identifies the polypeptide and/or the fragment asimmunogenic or associated with the autoimmune disorder or autoimmuneresponse in the subject.

30. The method of any one of items 1 to 12 further comprising predictingwhether the subject will have a clinical response to administration of acheckpoint inhibitor to treat cancer, the method comprising determiningwhether one or more cancer associated antigens together comprise atleast two different amino acid sequences each of which is a T cellepitope capable of binding to at least three HLA class I of the subjectand predicting

-   -   that the subject will have a clinical response to administration        of a checkpoint inhibitor if the one or more cancer associated        antigens together comprise at least two different sequences each        of which is a T cell epitope capable of binding to at least        three HLA class I molecules of the subject; or    -   B. that the subject will not have a clinical response to        administration of a checkpoint inhibitor if the one or more        cancer associated antigens together comprise no more than one        sequence that is a T cell epitope capable of binding to at least        three HLA class I molecules of the subject.

31. The method of any one of items 1 to 12, further comprisingdetermining the likelihood that the subject will have a clinicalresponse to administration of a checkpoint inhibitor to treat cancer,the method comprising

-   -   (i) selecting a plurality of polypeptide antigens that are        associated with the cancer type of the subject;    -   (ii) identifying which of said cancer associated antigens        comprise an amino acid sequence that is a T cell epitope capable        of binding to at least three HLA class I molecules of the        subject; and    -   (iii) using population expression data for each cancer        associated antigen identified in step to determine the        likelihood that the subject will have a clinical response to        administration of a checkpoint inhibitor to treat cancer,        wherein a higher probability that the subject expresses one or        more of the cancer associated antigens identified in step (ii)        that together comprise at least two amino acid sequences each of        which is a T cell epitope capable of binding to at least three        HLA class I molecules of the subject corresponds to a more        likely clinical response.

32. The method of item 30 or item 31 further comprising selecting orrecommending administration of a checkpoint inhibitor for treatment ofthe subject.

33. The method of item 32 further comprising administering a checkpointinhibitor to the subject.

34. A method of treatment of a human subject in need thereof, the methodcomprising administering to the subject a checkpoint inhibitor, whereinthe subject has been predicted to respond, or to be likely to respond,to administration of a checkpoint inhibitor by a method according toitem 30 or item 31.

35. The method of any one of items 13, 15 to 18 and 30, wherein thesubject has been predicted to have a toxic immune response or ADAdevelopment, or not to have a cytotoxic T cell or helper T cell orclinical response, or not to respond to treatment with a checkpointinhibitor and the method further comprises selecting or recommending adifferent treatment for the subject.

36. A method of designing or preparing a human subject-specificpharmaceutical composition or kit or panel of polypeptides for use in amethod of treatment of a specific human subject, the method comprising:

-   -   (i) selecting a fragment of a polypeptide, which fragment has        been identified as immunogenic for the subject by the method of        any one of items 2 to 11;    -   (ii) if the fragment selected in step (i) is an HLA class        I-binding epitope, optionally selecting a longer fragment of the        polypeptide, which longer fragment        -   a. comprises the fragment selected in step (i); and        -   b. is a T cell epitope capable of binding at least three or            to the most possible HLA class II molecules of the subject;    -   (iii) selecting a first sequence of up to 50 consecutive amino        acids of the polypeptide, which consecutive amino acids comprise        the amino acid sequence of the fragment selected in step (i) or        the longer fragment selected in step (ii);    -   (iv) repeating steps (i) to (iii) to select a second amino acid        sequence of up to 50 consecutive amino acids of the same or a        different polypeptide to the first amino acid sequence;    -   (v) optionally further repeating steps (i) to (iii) to select        one or more additional amino acid sequences of up to 50        consecutive amino acids of the same or different polypeptides to        the first and second amino acid sequences; and    -   (vi) designing or preparing a subject-specific pharmaceutical        composition, kit or panel of polypeptides having as active        ingredients one or more polypeptides that together have all of        the amino acid sequences selected in the preceding steps,        optionally wherein one or more or each sequence is flanked at        the N and/or C terminus by additional amino acids that are not        part of the sequence of the polypeptides.

37. The method of item 36, wherein each polypeptide either consists ofone of the selected amino acid sequences, or comprises or consists oftwo or more of the selected amino acid sequences arranged end to end oroverlapping in a single peptide.

38. The method of item 37, wherein all of the neoepitopes formed at thejoin between any two of the selected amino acid sequences arranged endto end in a single polypeptide have been screened to eliminatepolypeptides comprising a neoepitope amino acid sequence that

-   -   (i) corresponds to a fragment of a human polypeptide expressed        in healthy cells;    -   (ii) is a T cell epitope capable of binding to at least two HLA        class I molecules of the subject; or    -   (iii) meets both requirements (i) and (ii).

39. The method of any of items 36 to 38, wherein the one or morepolypeptides have been screened to eliminate polypeptides comprising anamino acid sequence that

-   -   (i) corresponds to a fragment of a human polypeptide expressed        in healthy cells; or    -   (ii) corresponds to a fragment of a human polypeptide expressed        in healthy cells and is a T cell epitope capable of binding to        at least two HLA class I molecules of the subject.

40. A human subject-specific pharmaceutical composition, kit or panel ofpolypeptides for use in a method of inducing an immune response in aspecific human subject, and designed or prepared for the subjectaccording to the method of any one of items 36 to 39, wherein thecomposition or kit optionally comprises at least one pharmaceuticallyacceptable diluent, carrier, or preservative.

41. A human subject-specific pharmaceutical composition, kit or panel ofpolypeptides for use in a method of treatment of a specific humansubject in need thereof, the composition, kit or panel comprising asactive ingredients a first and a second peptide and optionally one ofmore additional peptides, wherein each peptide comprises an amino acidsequence that is a T cell epitope capable of binding to at least two HLAclass I molecules and/or at least two HLA class II molecules of thesubject, wherein the amino acid sequence of the T cell epitope of thefirst, second and optionally any additional peptides are different fromeach other, and wherein the pharmaceutical composition or kit optionallycomprises at least one pharmaceutically acceptable diluent, carrier, orpreservative.

42. A human subject-specific pharmaceutical composition, kit or panel ofpolypeptides for use in a method of treatment of a specific humansubject in need thereof, the composition or kit comprising as an activeingredient a polypeptide comprising a first region and a second regionand optionally one of more additional regions, wherein each regioncomprises an amino acid sequence that is a T cell epitope capable ofbinding to at least two HLA class I molecules and/or at least two HLAclass II molecules of the subject, wherein the amino acid sequence ofthe T cell epitope of the first, second and optionally any additionalregions are different from each other, and wherein the pharmaceuticalcomposition or kit optionally comprises at least one pharmaceuticallyacceptable diluent, carrier, or preservative.

43. The human subject-specific pharmaceutical composition, kit or panelof item 41 or item 42, wherein one or more or each of the peptides orregions comprises an amino acid sequence that is a T cell epitopecapable of binding to at least two HLA class I molecules of the subject.

44. The human subject-specific pharmaceutical composition, kit or panelof any one of items 41 to 43, wherein one or more or each of thepeptides or regions comprises an amino acid sequence that is a T cellepitope capable of binding to at least three HLA class I molecules ofthe subject.

45. The human subject-specific pharmaceutical composition, kit or panelof any one of items 41 to 44, wherein one or more or each of thepeptides or regions comprises an amino acid sequence that is a T cellepitope capable of binding to at least three HLA class II molecules ofthe subject.

46. The human subject-specific pharmaceutical composition, kit or panelof item 44 or item 45 wherein one or more or each of the peptides orregions comprises an amino acid sequence that is a T cell epitopecapable of binding at least one HLA class II molecule of the subject,wherein the HLA class II-binding T cell epitope comprises an amino acidsequence that is a T cell epitope capable of binding to at least two HLAclass I molecules of the subject.

47. The human subject-specific pharmaceutical composition, kit or panelof any one of items 41 to 46, wherein one or more or each of thepeptides or regions comprises a sequence of up to 50 consecutive aminoacids of a polypeptide that is expressed by a pathogenic organism, avirus or a cancer cell, is associated with an autoimmune disorder, or isan allergen, wherein the sequence comprises the T cell epitope of thepeptide or region that is capable of binding to at least two HLA class Ior class II molecules of the subject, optionally wherein one or more oreach of the polypeptide sequences is flanked at the N and/or C terminusby additional amino acids that are not part of the amino acid sequenceof the polypeptide(s).

48. The human subject-specific pharmaceutical composition, kit or panelof any one of items 41 to 47, wherein one or more of the polypeptide(s)are selected from the antigens listed in Tables 2 to 6.

49. The human subject-specific pharmaceutical composition, kit or panelof any one of items 41 to 48, wherein the polypeptide(s) are antigens orneoantigens expressed by a cancer cell, optionally wherein the cancercell is in a sample taken from the subject.

50. The human subject-specific pharmaceutical composition, kit or panelof any one of items 41 to 49, wherein the polypeptide(s) are mutationalneoantigen(s), optionally wherein the neoantigen(s) are present in asample obtained from the subject; and/or the T cell epitope(s) eachcomprise a neoantigen specific mutation.

51. The human subject-specific pharmaceutical composition, kit or panelof any one of items 47 to 50 wherein two or more or each of thepolypeptide sequences of up to 50 consecutive amino acids are fromdifferent polypeptides.

52. The human subject-specific pharmaceutical composition, kit or panelof any one of items 47 to 51, wherein one or more or each of thesequences of up to 50 consecutive amino acids comprises an amino acidsequence that

-   -   (a) comprises an amino acid sequence that is a T cell epitope        capable of binding to at least three HLA class I molecules of        the subject; and    -   (b) is a T cell epitope capable of binding to at least three HLA        class II molecules of the subject or to the most possible HLA        class II molecules of the subject for a sequence comprising the        HLA class I-binding epitope of (a).

53. The human subject-specific pharmaceutical composition, kit or panelof item 47 to 52 wherein one or more or each polypeptide either

-   -   (a) consists of one of said sequence of up to 50 consecutive        amino acids from a polypeptide that is expressed by a pathogenic        organism, a virus or a cancer cell, is associated with an        autoimmune disorder or is an allergen; or    -   (b) comprises or consist of two or more of said sequences of up        to 50 consecutive amino acids arranged end to end or overlapping        in a single peptide.

54. The human subject-specific pharmaceutical composition, kit or panelof item 53 wherein the one or more peptides do not comprise anyneoepitopes that span a join between any two of said amino acidsequences that are arranged end to end in a single peptide and that

-   -   (i) corresponds to a fragment of a human polypeptide expressed        in healthy cells;    -   (ii) is a T cell epitope capable of binding to at least two HLA        class I molecules of the subject; or    -   (iii) meets both requirements (i) and (ii).

55. The human subject-specific pharmaceutical composition, kit or panelof any of items 41 to 54 wherein the one or more polypeptides do notcomprise any amino acid sequences that

-   -   (i) corresponds to a fragment of a human polypeptide expressed        in healthy cells; or    -   (ii) corresponds to a fragment of a human polypeptide expressed        in healthy cells and is a T cell epitope capable of binding to        at least two HLA class I molecules of the subject.

56. A method of treatment comprising administering to a human subject inneed thereof a human subject-specific pharmaceutical composition or thepolypeptides of a kit or panel of polypeptides according to any one ofitems 41 to 55, wherein the pharmaceutical composition, kit or panel isspecific for the subject, optionally wherein the method is for thetreatment of cancer.

57. The method of treatment according to any one of items 28, 34 and 56wherein the treatment is administered in combination with chemotherapy,targeted therapy or a checkpoint inhibitor.

58. A method of designing or preparing a polypeptide for inducing animmune response in a specific human subject the method comprisingselecting an amino acid sequence that is a T cell epitope capable ofbinding to at least three HLA class I molecules or at least three HLAclass II molecules of the subject, and designing or preparing apolypeptide comprising the selected amino acid sequence.

59. The method of item 58, which is

-   -   (a) a method of designing or preparing a polypeptide for        inducing a cytotoxic T cell response in a specific human        subject, the method comprising selecting an amino acid sequence        that is a T cell epitope capable of binding to at least three        HLA class I molecules of the subject, and designing or preparing        a polypeptide comprising the selected amino acid sequence; or    -   (b) a method of designing or preparing a polypeptide for        inducing a helper T cell response, the method comprising        selecting an amino acid sequence that is a T cell epitope        capable of binding to at least three HLA class II molecules of        the subject, and designing or preparing a polypeptide comprising        the selected amino acid sequence.

60. The method of item 58 or claim 59 further comprising administeringthe polypeptide to the subject.

61. A method of inducing an immune response in a subject, the methodcomprising administering to the subject a polypeptide designed accordingto the method of item 58 or item 59.

62. A method of inducing an immune response in a specific human subject,the method comprising designing or preparing a peptide according to themethod of item 58 or item 59, and administering the peptide to thesubject.

63. A system comprising

-   -   (c) a method of designing or preparing a polypeptide for        inducing a cytotoxic T cell response in a specific human        subject, the method comprising selecting an amino acid sequence        that is a T cell epitope capable of binding to at least three        HLA class I molecules of the subject, and designing or preparing        a polypeptide comprising the selected amino acid sequence; or    -   (d) a method of designing or preparing a polypeptide for        inducing a helper T cell response, the method comprising        selecting an amino acid sequence that is a T cell epitope        capable of binding to at least three HLA class II molecules of        the subject, and designing or preparing a polypeptide comprising        the selected amino acid sequence.

64. The storage system of item 63 further comprising

-   -   (c) an output module configured to display        -   (i) a prediction of whether the one or more polypeptides is            immunogenic for the subject; or the sequence of one or more            fragments of the one or more polypeptides that are predicted            to be immunogenic for the subject;        -   (ii) a prediction of whether the individual will have an            immune response to administration of the one or more            polypeptides or one or more pharmaceutical compositions            comprising the one or more polypeptides as active            ingredients;        -   (iii) a prediction of whether the subject will have a            clinical response to a method of treatment comprising            administering to the subject one or more pharmaceutical            compositions comprising the one or more polypeptides as            active ingredients;        -   (iv) the likelihood that the subject will have a clinical            response to administration of one or more pharmaceutical            compositions comprising the one or more polypeptides as            active ingredients;        -   (v) a prediction of whether administration of the one or            more polypeptides or one or more pharmaceutical compositions            comprising the one or more polypeptides will induce a toxic            immune response in the subject;        -   (vi) a prediction that the one or more polypeptides is            associated with an autoimmune disorder in the subject;        -   (vii) a prediction of whether the subject will have a            clinical response to administration of a checkpoint            inhibitor;        -   (viii) a recommendation of whether or not the subject should            be treated by administration of the one or more polypeptides            and/or one or more pharmaceutical compositions.

EXAMPLES Example 1—HLA-Epitope Binding Prediction Process and Validation

Predicted binding between particular HLA and epitopes (9 mer peptides)was based on the Immune Epitope Database tool for epitope prediction(iedb.org).

The HLA I-epitope binding prediction process was validated by comparisonwith HLA I-epitope pairs determined by laboratory experiments. A datasetwas compiled of HLA I-epitope pairs reported in peer reviewedpublications or public immunological databases.

The rate of agreement with the experimentally determined dataset (Table9) was determined. The binding HLA I-epitope pairs of the dataset werecorrectly predicted with a 93% probability. Coincidentally thenon-binding HLA I-epitope pairs were also correctly predicted with a 93%probability.

TABLE 9 Analytical specificity and sensitivity of the HLA-epitopebinding prediction process. True epitopes (n = 327) False epitopes (n =100) HLA-epitope pairs (Binder match) (Non-binder match) HIV 91% (32)82% (14) Viral 100% (35) 100% (11) Tumor 90% (172) 94% (32) Other(fungi, bacteria, etc.) 100% (65) 95% (36) All 93% (304) 93% (93)

The accuracy of the prediction of multiple HLA binding epitopes wasdetermined. Based on the analytical specificity and sensitivity usingthe 93% probability for both true positive and true negative predictionand 7% (=100%-93%) probability for false positive and false negativeprediction, the probability of the existence of a multiple HLA bindingepitope in a person can be calculated. The probability of multiple HLAbinding to an epitope shows the relationship between the number of HLAsbinding an epitope and the expected minimum number of real binding. PerPEPI definition three is the expected minimum number of HLA to bind anepitope (bold).

TABLE 10 Accuracy of multiple HLA binding epitopes predictions. Expectedminimum number of real Predicted number of HLAs binding to an epitopeHLA binding 0 1 2 3 4 5 6 1 35% 95% 100% 100% 100% 100% 100% 2  6% 29% 90%  99% 100% 100% 100% 3  1%  4%  22%  84%  98% 100% 100% 4  0%  0% 2%  16%  78%  96%  99% 5  0%  0%  0%  1%  10%  71%  94% 6  0%  0%  0% 0%  0%  5%  65%

The validated HLA-epitope binding prediction process was used todetermine all HLA-epitope binding pairs described in the Examples below.

Example 2—Epitope Presentation by Multiple HLA Predicts Cytotoxic TLymphocyte (CTL) Response

The presentation of one or more epitopes of a polypeptide antigen by oneor more HLA I of an individual is predictive for a CTL response wasdetermined.

The study was carried out by retrospective analysis of six clinicaltrials, conducted on 71 cancer and 9 HIV-infected patients (Table11)¹⁻⁷. Patients from these studies were treated with an HPV vaccine,three different NY-ESO-1 specific cancer vaccines, one HIV-1 vaccine anda CTLA-4 specific monoclonal antibody (Ipilimumab) that was shown toreactivate CTLs against NY-ESO-1 antigen in melanoma patients. All ofthese clinical trials measured antigen specific CD8+ CTL responses(immunogenicity) in the study subjects after vaccination. In some cases,correlation between CTL responses and clinical responses were reported.

No patient was excluded from the retroactive study for any reason otherthan data availability. The 157 patient datasets (Table 11) wererandomized with a standard random number generator to create twoindependent cohorts for training and evaluation studies. In some casesthe cohorts contained multiple datasets from the same patient, resultingin a training cohort of 76 datasets from 48 patients and atest/validation cohort of 81 datasets from 51 patients.

TABLE 11 Summary of patient datasets # Data Immunoassay sets performedin Clinical Target # (#antigen × the clinical HLA genotyping trialImmunotherapy Antigen Disease Patients* #patient) trials** method Ref 1VGX-3100 HPV16-E6 Cervical 17/18 5 × 17 IFN-γ ELISPOT High Resolution 1HPV16-E7 cancer SBT HPV18-E6 HPV18-E7 HPV16/18 2 HIVIS vaccine HIV-1 GagAIDS  9/12 2 × 9 IFN-γ ELISPOT Low-Medium 2 HIV-1 RT Resolution SSO 3rNY-ESO-1 NY-ESO-1 Breast-and 18/18 1 × 18 In vitro and High Resolution3 ovarian Ex vivo IFN-γ SBT 4 cancers, ELISPOT melanoma and sarcoma 4Ipilimumab NY-ESO-1 Metastatic 19/20 1 × 19 ICS after T- Low to medium 5melanoma cell resolution stimulation typing, SSP of genomic DNA, highresolution sequencing 5 NY-ESO-1f NY-ESO-1 Esophageal-, 10/10 1 × 10 ICSafter T- SSO probing 6 (91-110) non-small- cell and SSP of cell lung-stimulation genomic DNA and gastric cancer 6 NY-ESO-1 NY-ESO-1Esophageal- 7/9 1 × 7 ICS after T- SSO probing 7 overlapping (79-173)and lung cell and SSP of peptides cancer, stimulation genomic DNAmalignant melanoma Total 6 7 80 157 N/A *Number of patients used in theretrospective analysis from the original number of patient of theclinical trials. **Immunoassays are based on T cell stimulation withantigen-specific peptide pools and quantify the released cytokines bydifferent techniques. CT: Clinical trial; SBT: Sequence Based Typing;SSO: Sequence-Specific Oligonucleotide; ICS: Intracellular cytokinestaining; SSP: Sequence-specific priming

The reported CTL responses of the training dataset were compared withthe HLA I restriction profile of epitopes (9 mers) of the vaccineantigens. The antigen sequences and the HLA I genotype of each patientwere obtained from publicly available protein sequence databases or peerreviewed publications and the HLA I-epitope binding prediction processwas blinded to patients' clinical CTL response data. The number ofepitopes from each antigen predicted to bind to at least 1 (PEPI1+), orat least 2 (PEPI2+), or at least 3 (PEPI3+), or at least 4 (PEPI4+), orat least 5 (PEPI5+), or all 6 (PEPI6) HLA class I molecules of eachpatient was determined and the number of HLA bound were used asclassifiers for the reported CTL responses. The true positive rate(sensitivity) and true negative rate (specificity) were determined fromthe training dataset for each classifier (number of HLA bound)separately.

ROC analysis was performed for each classifier. In a ROC curve, the truepositive rate (Sensitivity) was plotted in function of the falsepositive rate (1-Specificity) for different cut-off points (FIG. 1).Each point on the ROC curve represents a sensitivity/specificity paircorresponding to a particular decision threshold (epitope (PEPI) count).The area under the ROC curve (AUC) is a measure of how well theclassifier can distinguish between two diagnostic groups (CTL responderor non-responder).

The analysis unexpectedly revealed that predicted epitope presentationby multiple class I HLAs of a subject (PEPI2+, PEPI3+, PEPI4+, PEPI5+,or PEPI6), was in every case a better predictor of CTL response thanepitope presentation by merely one or more HLA class I (PEPI1+,AUC=0.48, Table 12).

TABLE 12 Determination of diagnostic value of the PEPI biomarker by ROCanalysis Classifiers AUC PEPI1+ 0.48 PEPI2+ 0.51 PEPI3+ 0.65 PEPI4+ 0.52PEPI5+ 0.5 PEPI6+ 0.5

The CTL response of an individual was best predicted by considering theepitopes of an antigen that could be presented by at least 3 HLA class Iof an individual (PEPI3+, AUC=0.65, Table 12). The threshold count ofPEPI3+ (number of antigen-specific epitopes presented by 3 or more HLAof an individual) that best predicted a positive CTL response was 1(Table 13). In other words, at least one antigen-derived epitope ispresented by at least 3 HLA class I of a subject (≥1 PEPI3+), then theantigen can trigger at least one CTL clone, and the subject is a likelyCTL responder. Using the ≥1 PEPI3+ threshold to predict likely CTLresponders (“≥1 PEPI3+ Test”) provided 76% diagnostic sensitivity (Table13).

TABLE 13 Determination of the ≥1 PEPI3+ threshold to predict likely CTLresponders in the training dataset. PEPI3+ Count 1 2 3 4 5 6 7 8 9 10 1112 Sensitivity: 0.76 0.60 0.31 0.26 0.14 0.02 0 0 0 0 0 0 1-Specificity:0.59 0.24 0.21 0.15 0.09 0.06 0.06 0.03 0.03 0.03 0.03 0.03

Example 3—Validation of the ≥1 PEPI3+ Test

The test cohort of 81 datasets from 51 patients was used to validate the≥1 PEPI3+ threshold to predict an antigen-specific CTL response. Foreach dataset in the test cohort it was determined whether the ≥1 PEPI3+threshold was met (at least one antigen-derived epitope presented by atleast three class I HLA of the individual). This was compared with theexperimentally determined CTL responses reported from the clinicaltrials (Table 14).

The clinical validation demonstrated that a PEPI3+ peptide induce CTLresponse in an individual with 84% probability. 84% is the same valuethat was determined in the analytical validation of the PEPI3+prediction, epitopes that binds to at least 3 HLAs of an individual(Table 10). These data provide strong evidences that immune responsesare induced by PEPIs in individuals.

TABLE 14 Diagnostic performance characteristics of the ≥1 PEPI3+ Test (n= 81). Performance characteristic Description Result Positive 100%[A/(A + B)] The likelihood that an individual that meets the ≥1 84%predictive PEPI3+ threshold has antigen-specific CTL value (PPV)responses after treatment with immunotherapy. Sensitivity 100% [A/(A +C)] The proportion of subjects with antigen-specific 75% CTL responsesafter treatment with immunotherapy who meet the ≥1 PEPI3+ threshold.Specificity 100% [D/(B + D)] The proportion of subjects withoutantigen-specific 55% CTL responses after treatment with immunotherapywho do not meet the ≥1 PEPI3+ threshold. Negative 100% [D/(C + D)] Thelikelihood that an individual who does not meet 42% predictive the ≥1PEPI3+ threshold does not have antigen- value (NPV) specific CTLresponses after treatment with immunotherapy. Overall 100% [(A + D)/N]The percentage of predictions based on the ≥1 70% percent PEPI3+threshold that match the experimentally agreement determined result,whether positive or negative. (OPA) Fisher's exact (p) 0.01

ROC analysis determined the diagnostic accuracy, using the PEPI3+ countas cut-off values (FIG. 2). The AUC value=0.73. For ROC analysis an AUCof 0.7 to 0.8 is generally considered as fair diagnostic.

A PEPI3+ count of at least 1 (≥1 PEPI3+) best predicted a CTL responsein the test dataset (Table 15). This result confirmed the thresholddetermined during the training (Table 12).

TABLE 15 Confirmation of the ≥1 PEPI3+ threshold to predict likely CTLresponders in the test/validation dataset. PEPI3+ Count 1 2 3 4 5 6 7 89 10 11 12 Sensitivity: 0.75 0.52 0.26 0.23 0.15 0.13 0.08 0.05 0 0 0 01-Specificity: 0.45 0.15 0.05 0 0 0 0 0 0 0 0 0

Example 4—The ≥1 PEPI3+ Test Predicts CD8+ CTL Reactivities

The ≥1 PEPI3+ Test was compared with a previously reported method forpredicting a specific human subject's CTL response to peptide antigens.

The HLA genotypes of 28 cervical cancer and VIN-3 patients that receivedthe HPV-16 synthetic long peptide vaccine (LPV) in two differentclinical trials were determined from DNA samples⁸ ⁸ ⁹ ¹⁰. The LPVconsists of long peptides covering the HPV-16 viral oncoproteins E6 andE7. The amino acid sequence of the LPV was obtained from thesepublications. The publications also report the T cell responses of eachvaccinated patient to pools of overlapping peptides of the vaccine.

For each patient epitopes (9 mers) of the LPV that are presented by atleast three patient class I HLA (PEPI3+s) were identified and theirdistribution among the peptide pools was determined. Peptides thatcomprised at least one PEPI3+ (≥1 PEPI3+) were predicted to induce a CTLresponse. Peptides that comprised no PEPI3+ were predicted not to inducea CTL response.

The ≥1 PEPI3+ Test correctly predicted 489 out of 512 negative CTLresponses and 8 out of 40 positive CTL responses measured aftervaccination (FIG. 3A). Overall, the agreement between the ≥1 PEPI3+ Testand experimentally determined CD8+ T cell reactivity was 90% (p<0.001).

For each patient the distribution among the peptide pools of epitopesthat are presented by at least one patient class I HLA (≥1 PEPI1+, HLArestricted epitope prediction, prior art method) was also determined. ≥1PEPI1+ correctly predicted 116 out of 512 negative CTL responses and 37out of 40 positive CTL responses measured after vaccination (FIG. 3B).Overall, the agreement between the HLA restricted epitope prediction (≥1PEPI1+) and CD8+ T cell reactivity was 28% (not significant).

Example 5—Prediction of HLA Class II Restricted CD4+ Helper T CellEpitopes

The 28 cervical cancer and VIN-3 patients that received the HPV-16synthetic long peptide vaccine (LPV) in two different clinical trials(as detailed in Example 4) were investigated for CD4+ T helper responsesfollowing LPV vaccination (FIG. 4A-B). The sensitivity of the predictionof HLA class II restricted epitopes was 78%, since the State of Art toolpredicted 84 positive responses (positive CD4+ T cell reactivity to apeptide pool for a person's DP alleles) out of 107 (sensitivity=78%).The specificity was 22% since it could rule out 7 negative responses outof 31. Overall, the agreement between HLA-restricted class II epitopeprediction and CD4+ T cell reactivity was 66%, which was statisticallynot significant.

Example 6—the ≥1 PEPI3+ Test Predicts T Cell Responses to Full LengthLPV Polypeptides

Using the same reported studies as Examples 4 and 5, the ≥1 PEPI3+ Testwas used to predict patient CD8+ and CD4+ T cell responses to the fulllength E6 and E7 polypeptide antigens of the LPV vaccine. Results werecompared to the experimentally determined responses were reported. TheTest correctly predicted the CD8+ T cell reactivity (PEPI3+) of 11 outof 15 VIN-3 patients with positive CD8+ T cell reactivity test results(sensitivity 73%, PPV 85%) and of 2 out of 5 cervical cancer patients(sensitivity 40%, PPV 100%). The CD4+ T cell reactivities (PEPI4+) werecorrectly predicted 100% both of VIN-3 and cervical cancer patients(FIG. 5).

Class I and class II HLA restricted PEPI3+ count was also observed tocorrelate with the reported clinical benefit to LPV vaccinated patients.Patients with higher PEPI3+ counts had either complete or partialresponse already after 3 months.

Example 7—Case Study

pGX3001 is an HPV16 based DNA vaccine containing full length E6 and E7antigens with a linker in between. pGX3002 is an HPV18 based DNA vaccinecontaining full length E6 and E7 antigens with a linker in between. APhase II clinical trial investigated the T cell responses of 17HPV-infected patients with cervical cancer who were vaccinated with bothpGX3001 and pGX3002 (VGX-3100 vaccination)¹.

FIGS. 5A-D and FIG. 6 shows for two illustrative patients (patient 12-11and patient 14-5) the position of each epitope (9 mer) presented by atleast 1 (PEPI1+), at least 2 (PEPI2+), at least 3 (PEPI3+), at least 4(PEPI4+), at least 5 (PEPI5+), or all 6 (PEPI6) class I HLA of thesepatients within the full length sequence of the two HPV-16 and twoHPV-18 antigens.

Patient 12-11 had an overall PEPI1+ count of 54 for the combinedvaccines (54 epitopes presented by one or more class I HLA). Patient14-5 had a PEPI1+ count of 91. Therefore patient 14-5 has a higherPEPI1+ count than patient 12-11 with respect to the four HPV antigens.The PEPI1+s represent the distinct vaccine antigen specific HLArestricted epitope sets of patients 12-11 and 14-5. Only 27 PEPI1+s werecommon between these two patients.

For the PEPI3+ counts (number of epitopes presented by three or morepatient class I HLA), the results for patients 12-11 and 14-5 werereversed. Patient 12-11 had a PEPI3+ count of 8, including at least onePEPI3+ in each of the four HPV16/18 antigens. Patient 14-5 had a PEPI3+count of 0.

The reported immune responses of these two patients matched the PEPI3+counts, not the PEPI1+ counts. Patient 12-11 developed immune responsesto each of the four antigens post-vaccination as measured by ELISpot,whilst patient 14-5 did not develop immune responses to any of the fourantigens of the vaccines. A similar pattern was observed when the PEPI1+and PEPI3+ sets of all 17 patients in the trial were compared. There wasno correlation between the PEPI1+ count and the experimentallydetermined T cell responses reported from the clinical trial. However,correlation between the T cell immunity predicted by the ≥1 PEPI3+ Testand the reported T cell immunity was observed. The ≥1 PEPI3+ Testpredicted the immune responders to HPV DNA vaccine.

Moreover, the diversity of the patient's PEPI3+ set resembled thediversity of T cell responses generally found in cancer vaccine trials.Patients 12-3 and 12-6, similar to patient 14-5, did not have PEPI3+spredicting that the HPV vaccine could not trigger T cell immunity. Allother patients had at least one PEPI3 predicting the likelihood that theHPV vaccine can trigger T cell immunity. 11 patients had multiple PEPI3+predicting that the HPV vaccine likely triggers polyclonal T cellresponses. Patients 15-2 and 15-3 could mount high magnitude T cellimmunity to E6 of both HPV, but poor immunity to E7. Other patients 15-1and 12-11 had the same magnitude response to E7 of HPV18 and HPV16,respectively.

Example 8—Design of a Model Population for Conducting in Silico Trialsand Identifying Candidate Precision Vaccine Targets for Large Population

An in silico human trial cohort of 433 subjects with complete 4-digitHLA class I genotype (2×HLA-A*xx:xx; 2×HLA-B*xx:xx; 2×HLA-C*xx:xx) anddemographic information. This Model Population has subjects with mixedethnicity having a total of 152 different HLA alleles that arerepresentative for ≥85% of presently known allele G-groups.

A database of a “Big Population” containing 7,189 subjects characterizedwith 4-digit HLA genotype and demographic information was alsoestablished. The Big Population has 328 different HLA class I alleles.The HLA allele distribution of the Model Population significantlycorrelated with the Big Population (Table 16) (Pearson p<0.001).Therefore the 433 patient Model Population is representative for a 16times larger population.

The Model Population is representative for 85% of the human race asgiven by HLA diversity as well as HLA frequency.

TABLE 16 Statistical analysis of HLA distributions in “Model Population”vs. “Big Population”. Group Group Pearson R name 1 name 2 valueCorrelation P Value 433 Model 7,189 Big 0.89 Strong P < 0.001 PopulationPopulation

Example 9—in Silico Trials Based on the Identification of Multiple HLABinding Epitopes Predict the Reported T Cell Response Rates of ClinicalTrials

The objective of this study was to determine whether a model population,such as the one described in Example 8, may be used to predict CTLreactivity rates of vaccines, i.e. used in an in silico efficacy trials.

Twelve peptide vaccines derived from cancer antigens that induced T cellresponses in a subpopulation of subjects were identified from peerreviewed publications. These peptides have been investigated in clinicaltrials enrolling a total of 172 patients (4 ethnicities). T cellresponses induced by the vaccine peptides have been determined fromblood specimens and reported. The immune response rate as the percentageof study subjects with positive T cell responses measured in theclinical trials was determined (FIG. 7).

TABLE 17 Clinical trials conducted with peptide vaccines. SEQ SourcePeptide Pop. Peptide vaccines ID NO antigen length T cell assay (n)Ethnicity Ref. MMNLMQPKTQQTYTYD 1 JUP 16 mer Multimer 18 Canadian 12staining GRGSTTTNYLLDRDDYRNTSD 2 ADA17 21 mer Multimer 18 Canadian 12staining LKKGAADGGKLDGNAKLNRSLK 3 BAP31 22 mer Multimer 18 Canadian 12staining FPPKDDHTLKFLYDDNQRPYPP 4 TOP2A 22 mer Multimer 18 Canadian 12staining RYRKPDYTLDDGHGLLRFKST 5 Abl-2 21 mer Multimer 18 Canadian 12staining QRPPFSQLHRFLADALNT 6 DDR1 18 mer Multimer 18 Canadian 12staining ALDQCKTSCALMQQHYDQTSCFSSP 7 ITGB8 25 mer Multimer 18 Canadian12 staining STAPPAHGVTSAPDTRPAPGSTAPP 8 MUC-1 25 mer Proliferation 80Canadian 13 YLEPGPVTA 9 gp100  9 mer Tetramer 18 US 14MTPGTQSPFFLLLLLTVLTVV 10 MUC-1 21 mer Cytotoxicity 10 Israeli 15SSKALQRPV 11 Bcr-Abl  9 mer ELISPOT 4 US 16 RMFPNAPYL 12 WT-1  9 merMultimer 24 US 17 staining RMFPNAPYL (HLA-A*0201) 13 WT-1  9 merCytokine 18 CEU 18 staining

The 12 peptides were investigated with the ≥1 PEPI3+ Test in each of the433 subjects of the Model Population described in Example 8. The “≥1PEPI3+ Score” for each peptide was calculated as the proportion ofsubjects in the Model Population having at least one vaccine derivedepitope that could bind to at least three subject-specific HLA class I(≥1 PEPI3+). If the corresponding clinical trial stratified patients forHLA allele selected population, the Model Population was also filteredfor subjects with the respective allele(s) (Example: WT1, HLA-A*0201).

The experimentally determined response rates reported from the trialswere compared with the ≥1 PEPI3+ Scores. The Overall Percentage ofAgreements (OPA) were calculated on the paired data (Table 18). We alsofound a linear correlation between ≥1 PEPI3+ Score and response rate(R²=0.77) (FIG. 7). This result shows that the identification ofpeptides predicted to bind to multiple HLAs of an individual is usefulto predict in silico the outcome of clinical trials.

TABLE 18Comparison of ≥1 PEPI3+ Scores and CTL response rates of 12 peptide vaccines.≥1PEPI3+ Score* SEQ Source Response rate (Model Peptide vaccine ID NOantigen (Clinical Trials) Population) OPA MMNLMQPKTQQTYTYD 1 JUP 0% 22%NA GRGSTTTNYLLDRDDYRNTSD 2 ADA17 11% 18% 61% LKKGAADGGKLDGNAKLNRSLK 3BAP31 11% 7% 64% FPPKDDHTLKFLYDDNQRPYPP 4 TOP2A 11% 39% 28%RYRKPDYTLDDGHGLLRFKST 5 Abl-2 17% 12% 71% QRPPFSQLHRFLADALNT 6 DDR1 17%5% 29% ALDQCKTSCALMQQHYDQTSCFSSP 7 ITGB8 28% 31% 90%STAPPAHGVTSAPDTRPAPGSTAPP 8 MUC-1 20% 2% 10% YLEPGPVTA 9 gp100 28% 4%14% MTPGTQSPFFLLLLLTVLTVV 10 MUC-1 90% 95% 95% SSKALQRPV 11 Bcr- 0% 0%100% Abl RMFPNAPYL 12 WT-1 100% 78% 78% RMFPNAPYL (HLA-A*0201) 13 WT-181% 61% 75% *% subjects in the Model Population with ≥1 vaccine derivedPEPI3+ 

Example 10. In Silico Trials Based on the Identification of Multiple HLABinding Epitopes Predict the Reported T Cell Response Rates of ClinicalTrials II

Nineteen clinical trials with published immune response rates (IRR)conducted with peptide or DNA based vaccines were identified (Table 19).These trials involved 604 patients (9 ethnicities) and covered 38vaccines derived from tumor and viral antigens. Vaccine antigen specificCTL responses were measured in each study patient and the response ratein the clinical study populations was calculated and reported.

Each vaccine peptide of the 19 clinical trials was investigated with the≥1 PEPI3+ Test in each subject of the Model Population. The ≥1 PEPI3+Score for each peptide was calculated as the proportion of subjects inthe Model Population having at least one vaccine derived PEPI3+. Theexperimentally determined response rates reported from the trials werecompared with the PEPI Scores, as in Example 9 (Table 20). A linearcorrelation between the response rate and ≥1 PEPI3+ Score (R²=0.70) wasobserved (FIG. 8). This result confirms that the identification ofpeptides predicted to bind to multiple HLAs of an individual can predictT cell responses of subjects, and in silico trials can predict theoutcome of clinical trials.

TABLE 19 Response rates published in clinical trials. Immunotherapy TypeCTL assay Pop. (n) Race/ Ethnicity Ref. StimuVax peptide Proliferation80 Canadian 13 gp100 vaccine DNA Tetramer 18 US 14 IMA901 phase Ipeptide ELISPOT 64 CEU 19 IMA901 phase II peptide Multimer 27 CEUstaining ICT107 peptide ICC 15 US 20 ProstVac DNA ELISPOT 32 CEU 87%, 21Afr. Am. 12%, Hisp. 1% Synchrotope TA2M DNA Tetramer 26 US 22 MELITAC12.1 peptide ELISPOT 167 US 23 WT1 vaccine peptide Tetramer 22 Japanese24 Ipilimumab (NY-ESO-1) Check-point ICC 19 US 5 inhibitor** VGX-3100DNA ELISPOT 17 US 1 HIVIS-1 DNA ELISPOT 12 CEU 98%, 2 Asian 1%, Hisp. 1%ImMucin peptide Cytotoxicity 10 Israeli 15 NY-ESO-1 OLP peptideIFN-gamma 7 Japanese 7 GVX301 peptide Proliferation 14 CEU 25 1 vaccinepeptide ELISPOT 12 US 26 WT1 vaccine peptide ICC 18 CEU 18 DPX-0907*peptide Multimer 18 Canadian 12 staining Melanoma peptide peptideELISPOT 26 White 27 vaccine

TABLE 20 Linear correlation between PEPI Score and response rate (R² =0.7). Clinical Trial ≥1 PEPI3+ Immunotherapy Response Rate Score* OPAStimuVax  20%  2% 10% (failed to show efficacy in Phase III) gp100vaccine  28%  4% 14% IMA901 phase I  74% 48% 65% IMA901 phase II  64%48% 75% ICT107  33% 52% 63% ProstVac  45% 56% 80% Synchrotope TA2M  46%24% 52% MELITAC 12.1  49% 47% 96% WT1 vaccine  59% 78% 76% Ipilimumab(NY-ESO-1*)  72% 84% 86% VGX-3100  78% 87% 90% HIVIS-1  80% 93% 86%ImMucin  90% 95% 95% NY-ESO-1 OLP 100% 84% 84% GVX301  64% 65% 98% WT1vaccine  83% 80% 96% WT1 vaccine  81% 61% 75% DPX-0907  61% 58% 95%Melanoma peptide vaccine  52% 42% 81% *% subjects in the ModelPopulation with ≥ 1 vaccine derived PEPI3+

Example 11—in Silico Trial Based on the Identification of Multiple HLABinding Epitopes in a Multi-Peptide Vaccine Predict the ReportedClinical Trial Immune Response Rate

IMA901 is a therapeutic vaccine for renal cell cancer (RCC) comprising 9peptides derived from tumor-associated peptides (TUMAPs) that arenaturally presented in human cancer tissue. A total of 96 HLA-A*02+subjects with advanced RCC were treated with IMA901 in two independentclinical studies (phase I and phase II). Each of the 9 peptides ofIMA901 have been identified in the prior art as HLA-A2-restrictedepitopes. Based on currently accepted standards, they are all strongcandidate peptides to boost T cell responses against renal cancer in thetrial subjects, because their presence has been detected in renal cancerpatients, and because the trial patients were specifically selected tohave at least one HLA molecule capable of presenting each of thepeptides.

For each subject in the Model population how many of the nine peptidesof the IMA901 vaccine were capable of binding to three or more HLA wasdetermined Since each peptide in the IMA901 vaccine is a 9 mer thiscorresponds to the PEPI3+ count. The results were compared with theimmune response rates reported for the Phase I and Phase II clinicaltrials (Table 21).

TABLE 21 Immune Response Rates in the Model Population and in twoclinical trials to IMA901 Model Population Immune responses (HLA-A2+)Phase I Phase II to TUMAPs (n = 180) (n = 27)* (n = 64)* No peptide 39%25% 36% 1 peptide 34% 44% 38% ≥2 peptides 27% 29% 26% (MultiPEPI Score)≥3 peptides  3% ND  3% *No of patients evaluated for immune responses

The phase I and phase II study results show the variability of theimmune responses to the same vaccine in different trial cohorts.Overall, however, there was a good agreement between response ratespredicted by the ≥2 PEPI3+ Test and the reported clinical responserates.

In a retrospective analysis, the clinical investigators of the trialsdiscussed above found that subjects who responded to multiple peptidesof the IMA901 vaccine were significantly (p=0.019) more likely toexperience disease control (stable disease, partial response) thansubjects who responded only to one peptide or had no response. 6 of 8subjects (75%) who responded to multiple peptides experienced clinicalbenefit in the trial, in contrast to 14% and 33% of 0 and 1 peptideresponders, respectively. The randomized phase II trial confirmed thatimmune responses to multiple TUMAPs were associated with a longeroverall survival.

Since the presence of PEPIs accurately predicted responders to TUMAPs,clinical responders to IMA901 are likely patients who can present ≥2PEPIs from TUMAPs. This subpopulation is only 27% of HLA-A*02 selectedpatients, and according to the clinical trial result, 75% of thissubpopulation is expected to experience clinical benefit. The sameclinical results suggest that 100% of patients would experience clinicalbenefit if patient selection is based on ≥3 PEPIs from TUMAPs, albeitthis population would represent only 3% of the HLA-A*02 selected patientpopulation. These results suggest that the disease control rate (stabledisease or partial response) is between 3% and 27% in the patientpopulation which was investigated in the IMA901 clinical trials. In theabsence of complete response, only a portion of these patients canexperience survival benefit.

These findings explain the absence of improved survival in the Phase IIIIMA901 clinical trial. These results also demonstrated that HLA-A*02enrichment of the study population was not sufficient to reach theprimary overall survival endpoint in the Phase III IMA901 trial. As theIMA901 trial investigators noted, there is a need for the development ofa companion diagnostic (CDx) to select likely responders to peptidevaccines. These findings also suggest that selection of patients with ≥2TUMAP specific PEPIs may provide sufficient enrichment to demonstratesignificant clinical benefit of IMA901.

Example 12—in Silico Trial Based on the Identification ofVaccine-Derived Multiple HLA Binding Epitopes Predict ReportedExperimental Clinical Response Rates

A correlation between the ≥2 PEPI3+ Score of immunotherapy vaccinesdetermined in the Model Population described in Example 8 and thereported Disease Control Rate (DCR, proportion of patients with completeresponses and partial responses and stable disease) determined inclinical trials was determined.

Seventeen clinical trials, conducted with peptide- and DNA-based cancerimmunotherapy vaccines that have published Disease Control Rates (DCRs)or objective response rate (ORR) were identified from peer reviewedscientific journals (Table 22). These trials involved 594 patients (5ethnicities) and covered 29 tumor and viral antigens. DCRs weredetermined according to the Response Evaluation Criteria in Solid Tumors(RECIST), which is the current standard for clinical trials, in whichclinical responses are based on changes in maximum cross-sectionaldimensions^(42,43, 44). In case there was no available DCR data,objective response rate (ORR) data was used, which is also definedaccording to the RECIST guidelines.

Table 23 compares the ≥2 PEPI3+ Score for each vaccine in the ModelPopulation and the published DCR or ORR. A correlation between thepredicted and measured DCR was observed providing further evidence thatnot only the immunogenicity but also the potency of cancer vaccinesdepends on the multiple HLA sequences of individuals (R2=0.76) (FIG. 9).

Assessment Pop. Study pop./ HLA Adm. Dose time Immuno-therapy AntigenSponsor Disease (n) Ethnicity restriction form (mg) Dosing schedule(weeks) Ref. IMA901 phase I 9 TAAs Immatics Renal cell cancer 28 CEU A02i.d. 0.4 8× in 10 wks 12 19 IMA901 phase II 9 TAAs Immatics Renal cellcancer 68 CEU A02 i.d 0.4 7× in 5 wks then 10× 3 24 19 wks

TABLE 22 Clinical trials selected for Disease Control Rate (DCR)prediction. Ipilimumab NY-ESO-1 MSKCC Melanoma 19 US no i.v. 0.3 4×every 3 wks 24 5 3 10 HPV-SLP* HPV-16 E6, Leiden VIN 20 CEU no s.c. 0.33× every 3 wks 12 9 E7 University HPV-SLP* Leiden HPV-related 5 CEU nos.c. 0.3 3× every 3 wks 12 (OR) 10 University cervical cancer gp100-2gp100 BMS Melanoma 136 US A*0201 s.c. 1 4× every 3 wks 12 28 peptides*Immucin Muc-1 VaxilBio Myeloma 15 Israeli no s.c. 0.1 6× every 2 wks 12** 29 StimuVax Muc-1 Merck NSCLC 80 Canadian no s.c. 1 8× wkly then12 13, 30 every 6 wks VGX-3100 HPV-16&18 Inovio HPV-related 125 US noi.m. 6 0, 4, 12 wks 36 31 cervical cancer TSPP peptide Thymidylate SienaCRC, NSCLC, 21 CEU no s.c. 0.1 3 × 3 wks 12 32 vaccine synthaseUniversity Gallbladder 0.2 carc., Breast-, 0.3 Gastric cancer KIF20A-66KIF20A Chiba Metastatic 29 Japanese A*2402 s.c. 1 2 cycles 1, 8, 15, 12(OR) 33 peptide Tokushukai pancreatic 3 22 days then vaccine* Hospitalcancer every 2 wks Peptide 3 TAAs Kumamoto HNSCC 37 Japanese A*2402 s.c.1 8× wkly then 12 34 vaccine* University every 4 wks 7-peptide cock- 7TAAs Kinki Metastatic 30 Japanese A*2402 s.c. 1 Cycles: 5× wkly 10 (OR)35 tail vaccine* University colorectal cancer then 1 wk rest GVX301*hTERT University Prostate and 14 Japanese A02 i.d. 0.5 1, 3, 5, 7, 14,21, 12 25 Genoa renal cancer 35, 63 days MAGE-A3 MAGE-A3 AbramsonMultiple 26 US no s.c. 0.3 14, 42, 90, 24 36 Trojan* Cancer myeloma 120,150 days Center PepCan HPV-16 E6 University CIN2/3 23 US no i.m 0.05 4 ×3 wks 24 37 of Arkansas 0.1 0.25 0.5 Melanoma Tyrosinase, UniversityMelanoma 26 US A1, A2 or s.c. 0.1 6 cycles: 0, 7, 14,  6 27 peptidegp100 of Virginia A3 28, 35, 42 days vaccine* *Montanide ISA51 VG asadjuvant **Disease response was assessed according to the InternationalMyeloma Working Group response criteria⁴⁵

TABLE 23 The Disease Control Rates (DCRs) and MultiPEPI Scores(predicted DCR) in 17 clinical trials. MultiPEPI Score OverallPercentage of Immunotherapy DCR (Predicted DCR) Agreement IMA901 phase I43% 27%  61% IMA901 phase II 22% 27%  81% Ipilimumab 60% 65%  92%HPV-SLP 60% 70%  86% HPV-SLP 62% 70%  89% gp100-2 peptides 15% 11%  73%Immucin 73% 59%  81% StimuVax  0%  0% 100% VGX-3100 50% 56%  89% TSPPpeptide vaccine 48% 31%  65% KIF20A-66 peptide 26%  7%  27% vaccinePeptide vaccine 27% 10%  37% 7-peptide cocktail 10%  9%  90% vaccineGVX301 29%  7%  24% MAGE-A3 Trojan 35% 10%  29% PepCan 52% 26%  50%Melanoma peptide 12%  6%  50% vaccine

Example 13—the Set of Multiple HLA Binding Peptides from Tumor AntigensPredicts Responders to the Checkpoint Inhibitor Immunotherapy Ipilmumab

Whether survival benefit of melanoma patients treated with thecheckpoint inhibitor Ipilimumab can be predicted by the number ofmelanoma-specific PEPI3+s that are potentially expressed in thepatient's tumor was determined.

Eighty melanoma associated antigens (TAAs) were identified from which apanel of PEPI3+s (IPI-PEPI panel: 627 PEPIs) that are shared byIpilimumab treated melanoma patients with a prolonged clinical benefitand are absent in those without a prolonged benefit was selected. ThesePEPI3+ define the specific T cells that are re-activated by Ipilimumabto attack the patient's tumor cells. Patients with certain HLA sequencesthat can present more melanoma-specific PEPIs have more T cellsre-activated by Ipilimumab and a higher chance to benefit fromIpilimumab immunotherapy.

The clinical benefit from Ipilimumab treatment for 160 patients fromfour independent clinical trial cohorts was determined. Two cohorts werefrom the trials CA184-007 (10 mg/kg Ipilimumab) and CA184-002 (3 mg/kgIpilimumab) and two cohorts from published clinical trials 10 mg/kg and3/mg/kg Ipilimumab datasets^(5, 38, 39).

Epitopes from 80 melanoma antigens restricted to all the 6 HLA class Iof each patient were predicted and the number of melanoma-specificPEPI3+s restricted to at least 3 class I HLAs of each patient (4,668PEPIs) was then computed. Each patient with at least one out of 627PEPIs qualified as responder. The IPI-PEPI panel predicts the overallsurvival of both 10 mg/kg and 3 mg/kg Ipilimumab. Results were highlysignificant and consistent in the four independent cohort (FIGS. 10A-D).

Example 14: Multiple HLA Binding Epitopes Define Patient MutationalNeoantigens

The capability of the PEPI3+ to identify neoantigens from mutations wasdetermined. PEPI3+s of 110 melanoma patients treated with Ipilimumab wasdetermined using published exome mutation data³⁹. From the exomemutation data, mutations in 9,502 antigens from the 110 patients (FIG.11A). Median nonsynonymous mutational load per sample was highlyvariable, 309 (29-4,738) in the clinical benefit cohort and 147(7-5,854) in the minimal or no clinical benefit cohort. Due to theirepitope prediction results these mutations had 211 (8-1950) and 56(2-3444) neoepitopes in the clinical benefit cohort and the minimal orno clinical benefit cohorts, respectively.

Mutational PEPI3+ neoepitopes from the published mutations weredetermined (FIG. 1lB and Table 24). These mutations resulted in median16 PEPIs and 6 PEPIs neoepitopes in clinical benefit cohort and theminimal or no clinical benefit cohorts, respectively.

Results show that PEPIs define the mutational neoantigens derived fromgenetically altered proteins expressed in an individual. Suchneoantigens are PEPI3+ peptides that capable to activate T cells in thepatient's body. If a genetic alteration occurs in the tumor cell of theindividual that creates a PEPI3+ then this PEPI3+ can induce T cellresponses. These PEPI3+ containing peptides could be included in a drug(e.g. vaccine, T cell therapy) to induce immune response against theindividual tumor.

TABLE 24 Mutational neoantigen prediction using PEPI Test: Analysisresults of Van Allen et al. and PEPI Test on 110 melanoma patients.Result obtained from PEPI Results published Test analyses (ValidatedParameters by Van Allen et al. epitopes and PEPIs) Clinical Minimal orno Clinical Minimal or no benefit clinical Benefit benefit clinicalbenefit Patients (n = 27) (n = 73) (n = 27) (n = 73) Median mutations555 281 — — Median nonsynonymous 309 147 — — mutations Median expressedmutational 198 — — antigens Median neoepitope (only 9mer) 211 56 130 50Recurrent neoepitopes 28 Not provided 10 76 Median PEPI neoepitopes — —16 6 Recurrent PEPI neoepitopes — — 1 5

Example 15 in Silico Trials Based on the Identification of Multiple HLABinding Epitopes Predict the Reported Cellular Immune Response Rates toa Vaccine Targeting a Mutational Antigen

The epidermal growth factor receptor variant III (EGFRvIII) is atumor-specific mutation broadly expressed in glioblastoma multiforme(GBM) and other neoplasms. The mutation comprises an in-frame deletionof 801 bp from the extracellular domain of the EGFR that splits a codonand yields a novel glycine at the fusion junction.^(1, 2) This mutationencodes a constitutively active tyrosine kinase that increases tumorformation and tumor cell migration and enhances resistance againstradiation and chemotherapy.^(3, 4, 5, 6, 7, 8, 9) This insertion resultsin a tumor-specific epitope which is not found in normal adult tissuesmaking EGFRvIII a suitable target candidate for antitumorimmunotherapy.¹⁰ Rindopepimut is a 13-amino-acid peptide vaccine(LEEKKGNYVVTDHC (SEQ ID NO: 87)) spanning the EGFRvIII mutation with anadditional C-terminal cysteine residue.¹¹

In a phase II clinical study, the peptide conjugated to keyhole limpethemocyanin (KLH) was administered to newly diagnosed EGFRvIII-expressingGBM patients. The first three vaccinations were given biweekly, starting4 weeks after the completion of radiation. Subsequent vaccines weregiven monthly until radiographic evidence of tumor progression or death.All vaccines were given intradermally in the inguinal region.Immunologic evaluation showed only 3 out of 18 patients developingcellular immune response assessed by DTH reaction test.

An in silico trial with the Model Population of 433 subjects withRindopepimut sequence was conducted. 4 out of 433 subjects had PEPI3+,confirming the low immunogenicity found in the phase II study (Table25).

TABLE 25 Results of clinical trial and in silico study RespondersResponse rate Clinical trial (Phase II) 3/18  16.6% In silico study(PEPI3+ Test) 4/433   1%

An HLA map of the Rindopepimut on the HLA alleles of the subjects in theModel Population (FIG. 12) illustrates that very few HLA-A and HLA-Calleles can bind the vaccine epitopes which explains the lack of PEPI3+in the in silico cohort.

In a recent phase III clinical study the ineffectiveness was furtherdemonstrated when 745 patients were enrolled and randomly assigned toRindopepimut and temozolomide (n=371) or control and temozolomide(n=374) arms.¹² The trial was terminated for ineffectiveness after theinterim analysis. The analysis showed no significant difference inoverall survival: median overall survival was 20.1 months (95% CI18.5-22.1) in the Rindopepimut group versus 20.0 months (18.1-21.9) inthe control group (HR 1.01, 95% CI 0.79-1.30; p=0.93).

REFERENCES FOR EXAMPLE 15

-   1 Bigner et al. Characterization of the epidermal growth factor    receptor in human glioma cell lines and xenografts. Cancer Res 1990;    50: 8017-22.-   2 Libermann et al. Amplification, enhanced expression and possible    rearrangement of EGF receptor gene in primary human brain tumours of    glial origin. Nature 1985; 313: 144-7.-   3 Chu et al. Receptor dimerization is not a factor in the signalling    activity of a transforming variant epidermal growth factor receptor    (EGFRvIII). Biochem J 1997; 324: 855-61.-   4 Batra et al. Epidermal growth factor ligand-independent,    unregulated, cell-transforming potential of a naturally occurring    human mutant EGFRvIII gene. Cell Growth Differ 1995; 6: 1251-9.-   5 Nishikawa et al. A mutant epidermal growth factor receptor common    in human glioma confers enhanced tumorigenicity. PNAS 1994; 91:    7727-31.-   6 Lammering et al. Inhibition of the type III epidermal growth    factor receptor variant mutant receptor by dominant-negative    EGFR-CD533 enhances malignant glioma cell radiosensitivity. Clin    Cancer Res 2004; 10: 6732-43.-   7 Nagane et al. A common mutant epidermal growth factor receptor    confers enhanced tumorigenicity on human glioblastoma cells by    increasing proliferation and reducing apoptosis. Cancer Res 1996;    56: 5079-86.-   8 Lammering et al. Radiation-induced activation of a common variant    of EGFR confers enhanced radioresistance. Radiother Oncol 2004; 72:    267-73.-   9 Montgomery et al. Expression of oncogenic epidermal growth factor    receptor family kinases induces paclitaxel resistance and alters    0-tubulin isotype expression. J Biol Chem 2000; 275: 17358-63.-   10 Humphrey et al. Anti-synthetic peptide antibody reacting at the    fusion junction of deletion-mutant epidermal growth factor receptors    in human glioblastoma. PNAS 1990; 87: 4207-11.-   11 Sampson et al. Immunologic Escape After Prolonged    Progression-Free Survival With Epidermal Growth Factor Receptor    Variant III Peptide Vaccination in Patients With Newly Diagnosed    Glioblastoma. J Clin Oncol 28:4722-4729.-   12 Weller at al. Rindopepimut with temozolomide for patients with    newly diagnosed, EGFRvIII-expressing glioblastoma (ACT IV): a    randomised, double-blind, international phase 3 trial. Lancet Oncol    2017; 18(10): 1373-1385.

Example 16. Multiple HLA Binding Peptides of Individuals can PredictImmune-Toxicity

Thrombopoietin (TPO) is a highly immunogenic protein drug causingtoxicity in many patients. EpiVax/Genentech used State of Art technologyto identify class II HLA restricted epitopes and found that the mostimmunogenic region of the TPO is located in the C-terminal end of TPO(US20040209324 A1).

According to the present discloure we defined the multiple class II HLAbinding epitopes (PEPI3+s) from TPO in 400 HLA class II genotyped USsubjects were determined. Most of the PEPI3+ peptides of theseindividuals located within the N terminal region of the TPO between1-165 amino acids. PEPI3+ were spopradically identified in some subjectsalso in the C terminal region. However, these results were differentfrom the State of Art.

The published literature confirmed the disclosed results, demonstratingexperimental proof for the immunotoxic region being located at theN-terminal end of TPO^(40, 41). Most individuals treated with TPO drugmade anti-drug antibodies (ADA) ADA against this region of the drug.These antibodies not only abolished the therapeutic effect of the drugbut also caused systemic adverse events, i.e. immune-toxicity, likeantibody-dependent cytotoxicity (ADCC) and complement-dependentcytotoxicity associated with thrombocytopenia, neutropenia and anemia.These data demonstrate that the identification of multiple HLA bindingpeptides of individuals predicts the immune-toxicity of TPO. Therefore,the disclosure is useful to identify the toxic immunogenic region ofdrugs, to identify subjects who likely experience immune-toxicity fromdrugs, to identify regions of a polypeptide drug that may be targeted byADAs, and to identify subjects who likely experience ADA.

Example 17 Personalised Immunotherapy Composition for Treatment ofOvarian Cancer

This example describes the treatment of an ovarian cancer patient with apersonalised immunotherapy composition, wherein the composition wasspecifically designed for the patient based on her HLA genotype based onthe disclosure described herein. This Example and Example 19 belowprovide clinical data to support the principals regarding binding ofepitopes by multiple HLA of a subject to induce a cytotoxic T cellresponse on which the present disclosure is based.

The HLA class I and class II genotype of metastatic ovarianadenocarcinoma cancer patient XYZ was determined from a saliva sample.

To make a personalized pharmaceutical composition for patient XYZthirteen peptides were selected, each of which met the following twocriteria: (i) derived from an antigen that is expressed in ovariancancers, as reported in peer reviewed scientific publications; and (ii)comprises a fragment that is a T cell epitope capable of binding to atleast three HLA class I of patient XYZ (Table 26). In addition, eachpeptide is optimized to bind the maximum number of HLA class II of thepatient.

TABLE 26 XYZ ovarian cancer patient's personalized vaccine SEQ MAX MAXTarget Antigen ID HLA HLA XYZ's vaccine Antigen Expression20 mer peptides NO class I class II P0001_P1 AKAP4 89%NSLQKQLQAVLQWIAASQFN 14 3 5 P0001_P2 BORIS 82% SGDERSDEIVLTVSNSNVEE 15 42 P0001_P3 SPAG9 76% VQKEDGRVQAFGWSLPQKYK 16 3 3 P0001_P4 OY-TES-1 75%EVESTPMIMENIQELIRSAQ 17 3 4 P0001_P5 SP17 69% AYFESLLEKREKTNFDPAEW 18 31 P0001_P6 WT1 63% PSQASSGQARMFPNAPYLPS 19 4 1 P0001_P7 HIWI 63%RRSIAGFVASINEGMTRWFS 20 3 4 P0001_P8 PRAME 60% MQDIKMILKMVQLDSIEDLE 21 34 P0001_P9 AKAP-3 58% ANSVVSDMMVSIMKTLKIQV 22 3 4 P0001_P10 MAGE-A4 37%REALSNKVDELAHFLLRKYR 23 3 2 P0001_P11 MAGE-A9 37% ETSYEKVINYLVMLNAREPI24 3 4 P0001_P12a MAGE-A10 52% DVKEVDPTGHSFVLVTSLGL 25 3 4 P0001_P12bBAGE 30% SAQLLQARLMKEESPVVSWR 26 3 2

Eleven PEPI3 peptides in this immunotherapy composition can induce Tcell responses in XYZ with 84% probability and the two PEPI4 peptides(POC01-P2 and POC01-P5) with 98% probability, according to thevalidation of the PEPI Test shown in Table 10. T cell responses target13 antigens expressed in ovarian cancers. Expression of these cancerantigens in patient XYZ was not tested. Instead the probability ofsuccessful killing of cancer cells was determined based on theprobability of antigen expression in the patient's cancer cells and thepositive predictive value of the ≥1 PEPI3+ Test (AGP count). AGP countpredicts the effectiveness of a vaccine in a subject: Number of vaccineantigens expressed in the patient's tumor (ovarian adenocarcinoma) withPEPI. The AGP count indicates the number of tumor antigens that vaccinerecognizes and induces a T cell response against the patient's tumor(hit the target). The AGP count depends on the vaccine-antigenexpression rate in the subject's tumor and the HLA genotype of thesubject. The correct value must be between 0 (no PEPI presented byexpressed antigen) and maximum number of antigens (all antigens areexpressed and present a PEPI).

The probability that patient XYZ will express one or more of the 12antigens is shown in FIGS. 13A-B. AGP95=5, AGP50=7.9, mAGP=100%, AP=13.

A pharmaceutical composition for patient XYZ may be comprised of atleast 2 from the 13 peptides (Table 26), because the presence in avaccine or immunotherapy composition of at least two polypeptidefragments (epitopes) that can bind to at least three HLA of anindividual (≥2 PEPI3+) was determined to be predictive for a clinicalresponse. The peptides are synthetized, solved in a pharmaceuticallyacceptable solvent and mixed with an adjuvant prior to injection. It isdesirable for the patient to receive personalized immunotherapy with atleast two peptide vaccines, but preferable more to increase theprobability of killing cancer cells and decrease the chance of relapse.

For treatment of patient XYZ the 12 peptides were formulated as 4×3/4peptide (POC01/1, POC01/2, POC01/3, POC01/4). One treatment cycle isdefined as administration of all 13 peptides within 30 days.

Patient History:

Diagnosis: Metastatic ovarian adenocarcinoma

Age: 51

Family anamnesis: colon and ovary cancer (mother) breast cancer(grandmother)

Tumor Pathology: BRCA1-185delAG, BRAF-D594Y, MAP2K1-P293S, NOTCHI-S2450N

-   -   2011: first diagnosis of ovarian adenocarcinoma; Wertheim        operation and chemotherapy; lymph node removal    -   2015: metastasis in pericardial adipose tissue, excised    -   2016: hepatic metastases    -   2017: retroperitoneal and mesenteric lymph nodes have        progressed; incipient peritoneal carcinosis with small        accompanying ascites

Prior Therapy:

-   -   2012: Paclitaxel-carboplatin (6×)    -   2014: Caelyx-carboplatin (1×)    -   2016-2017 (9 months): Lymparza (Olaparib) 2×400 mg/day, oral    -   2017: Hycamtin inf. 5×2.5 mg (3× one seria/month)    -   PIT vaccine treatment began on 21 Apr. 2017.

TABLE 27 Patient XYZ peptide treatment schedule Vaccinations Lot #1^(st) cycle 2^(nd) cycle 3^(rd) cycle 4^(th) cycle POC01/1 N1727 21Apr. 2017 16 Jun. 2017 30 Aug. 2017 19 Oct. 2017 POC01/2 N1728 28 Apr.2017 31 May 2017 POC01/3 N1732 16 Jun. 2017 2 Aug. 2017 20 Sep. 2017POC01/4 N1736 15 May 2017 6 Jul. 2017

Patient' Tumor MRI Findings (Baseline Apr. 15, 2016)

-   -   Disease was confined primarily to liver and lymph nodes. The use        of MRI limits detection of lung (pulmonary) metastasis    -   May 2016-January 2017: Olaparib treatment    -   Dec/25/2016 (before PIT vaccine treatment) There was dramatic        reduction in tumor burden with confirmation of response obtained        at FU2    -   January-March 2017—TOPO protocol (topoisomerase)    -   April/6/2017 FU3 demonstrated regrowth of existing lesions and        appearance of new lesions leading to disease progression    -   Apr. 21 2017 START PIT    -   Jul/21/17 (after the 2^(nd) Cycle of PIT) FU4 demonstrated        continued growth in lesions and general enlargement of pancreas        and abnormal para pancreatic signal along with increased ascites    -   Jul/26/17—CBP+Gem+Avastin    -   Sep/20/17 (after 3 Cycles of PIT) FU5 demonstrated reversal of        lesion growth and improved pancreatic/parapancreatic signal. The        findings suggest pseudo progression    -   Nov 28/17 (after 4 Cycles of PIT) FU6 demonstrated best response        with resolution of non target lesions        MRI data for patient XYZ is shown in Table 28 and FIG. 14.

TABLE 28 Summary Table of Lesions Responses FU1 FU2 FU3 FU4 FU5 Lesion/Baseline (% Δ (% Δ (% Δ (% Δ (% Δ FU6 Best PD Time (% Δ from from fromfrom from from (% Δ from Response Time Point BL) BL) BL) BL) BL) BL) BL)Cycle Point TL1 NA −56.1 −44.4 −44.8 +109.3 −47.8 −67.3 FU6 FU4 TL2 NA−100.0 −100.0 −47.1 −13.1 −100.0 −100.0 FU1 FU3 TL3 NA −59.4 −62.3 −62.0−30.9 −66.7 −75.9 FU6 FU4 TL4 NA −65.8 −100.0 −100.0 −100.0 −100.0−100.0 FU2 NA SUM NA −66.3 −76.0 −68.9 −23.5 −78.2 −85.2 FU6 FU4

Example 18 Design of Personalised Immunotherapy Composition forTreatment of Breast Cancer

The HLA class I and class II genotype of metastatic breast cancerpatient ABC was determined from a saliva sample. To make a personalizedpharmaceutical composition for patient ABC twelve peptides wereselected, each of which met the following two criteria: (i) derived froman antigen that is expressed in breast cancers, as reported in peerreviewed scientific publications; and (ii) comprises a fragment that isa T cell epitope capable of binding to at least three HLA class I ofpatient ABC (Table 29). In addition, each peptide is optimized to bindthe maximum number of HLA class II of the patient. The twelve peptidestarget twelve breast cancer antigens. The probability that patient ABCwill express one or more of the 12 antigens is shown in FIG. 15.

TABLE 29 12 peptides for ABC breast cancer patient SEQ BRC09 vaccineTarget Antigen ID MAXHLA MAXHLA peptides Antigen Expression20 mer peptide NO Class I Class II PBRC01_cP1 FSIP1 49%ISDTKDYFMSKTLGIGRLKR 27 3 6 PBRC01_cP2 SPAG9 88% FDRNTESLFEELSSAGSGLI 283 2 PBRC01_cP3 AKAP4 85% SQKMDMSNIVLMLIQKLLNE 29 3 6 PBRC01_cP4 BORIS71% SAVFHERYALIQHQKTHKNE 30 3 6 PBRC01_cP5 MAGE-A11 59%DVKEVDPTSHSYVLVTSLNL 31 3 4 PBRC01_cP6 NY-SAR-35 49%ENAHGQSLEEDSALEALLNF 32 3 2 PBRC01 cP7 HOM-TES- 47% MASFRKLTLSEKVPPNHPSR33 3 5 85 PBRC01_cP8 NY-BR-1 47% KRASQYSGQLKVLIAENTML 34 3 6 PBRC01_cP9MAGE-A9 44% VDPAQLEFMFQEALKLKVAE 35 3 8 PBRC01_cP10 SCP-1 38%EYEREETRQVYMDLNNNIEK 36 3 3 PBRC01_cP11 MAGE-A1 37% PEIFGKASESLQLVFGIDVK37 3 3 PBRC01_cP12 MAGE-C2 21% DSESSFTYTLDEKVAELVEF 38 4 2Predicted efficacy: AGP95=4; 95% likelihood that the PIT Vaccine inducesCTL responses against 4 CTAs expressed in the breast cancer cells ofBRC09. Additional efficacy parameters: AGP50=6.3, mAGP=100%, AP=12.Detected efficacy after the 1^(st) vaccination with all 12 peptides: 83%reduction of tumor metabolic activity (PET CT data).

For treatment of patient ABC the 12 peptides were formulated as 4×3peptide (PBRO1/1, PBRO1/2, PBRO1/3, PBRO1/4). One treatment cycle isdefined as administration of all 12 different peptide vaccines within 30days.

Patient History

Diagnosis: bilateral metastatic breast carcinoma: Right breast is ERpositive, PR negative, Her2 negative; Left Breast is ER, PR and Her2negative.First diagnosis: 2013 (4 years before PIT vaccine treatment) 2016:extensive nietastatic disease with nodal involvement both above andbelow the diaphragm.Multiple liver and puinonar metastases.2016-2017 treatment: Etrozole, Ibrance (Palbociclib) and Zoladex

Results

Mar. 7, 2017: Prior PIT Vaccine treatmentHepatic multi-metastatic disease with truly extrinsic compression of theorigin of the choledochal duct and massive dilatation of the entireintrahepatic biliary tract. Celiac, hepatic hilar and retroperitonealadenopathyMay 26 2017: After 1 cycle of PITDetected efficacy: 83% reduction of tumor metabolic activity (PET CT)liver, lung lymph nodes and other metastases. Detected safety: SkinreactionsLocal inflammation at the site of the injections within 48 hoursfollowing vaccine administrations

Follow Up:

BRC-09 was treated with 5 cycles of PIT vaccine. She was feeling verywell and she refused a PET CT examination in September 2017. In Novembershe had symptoms, PET CT scan showed progressive disease, but sherefused all treatments. In addition, her oncologist found out that shedid not take Palbocyclib since spring/summer. Patient ABC passed away inJanuary 2018.

The combination of pablocyclib and the personalised vaccine was likelyto have been responsible for the remarkable early response observedfollowing administration of the vaccine. Palbocyclib has been shown toimprove the activity of immunotherapies by increases CTA presentation byHLAs and decreasing the proliferation of Tregs: (Goel et al. Nature.2017:471-475). The PIT vaccine may be used as add-on to the state-of-arttherapy to obtain maximal efficacy.

Example 19—Personalised Immunotherapy Composition for Treatment ofPatient with Late Stage Metastatic Breast Cancer

Patient BRC05 was diagnosed with inflammatory breast cancer on the rightwith extensive lymphangiosis carcinomatose. Inflammatory breast cancer(IBC) is a rare, but aggressive form of locally advanced breast cancer.It's called inflammatory breast cancer because its main symptoms areswelling and redness (the breast often looks inflamed). Mostinflammatory breast cancers are invasive ductal carcinomas (begin in themilk ducts). This type of breast cancer is associated with theexpression of oncoproteins of high risk Human Papilloma Virus. Indeed,HPV16 DNA was diagnosed in the tumor of this patient.

Patient's stage in 2011 (6 years prior to PIT vaccine treatment)T4: Tumor of any size with direct extension to the chest wall and/or tothe skin (ulceration or skin nodules)pN3a: Metastases in ≥10 axillary lymph nodes (at least 1 tumordeposit >2.0 mm); or metastases to the infraclavicular (level IIIaxillary lymnph) nodes.

14 vaccine peptides were designed and prepared for patient BRC05 (Table30). Peptides PBRC05-PO1-P10 were made for this patient based onpopulation expression data. The last 3 peptides in the Table 29 (SSX-2,MORC, MAGE-B1) were designed from antigens that expression was measureddirectly in the tumor of the patient.

TABLE 30 Vaccine peptides for patient BRC05 BRC05 vaccine Target AntigenMAXHLA MAXHLA peptides Antigen Expression 20mer peptide Class I Class IIPBRC05_P1 SPAG9 88% XXXXXXXXXXXXXXXXXXXX 3 4 PBRC05_P2 AKAP4 85%XXXXXXXXXXXXXXXXXXXX 3 4 PBRC05_P3 MAGE-A11 59% XXXXXXXXXXXXXXXXXXXX 3 3PBRC05_P4 NY-SAR-35 49% XXXXXXXXXXXXXXXXXXXX 3 3 PBRC05_P5 FSIP1 49%XXXXXXXXXXXXXXXXXXXX 3 3 PBRC05_P6 NY-BR-1 47% XXXXXXXXXXXXXXXXXXXX 3 4PBRC05_P7 MAGE-A9 44% XXXXXXXXXXXXXXXXXXXX 3 3 PBRC05_P8 SCP-1 38%XXXXXXXXXXXXXXXXXXXX 3 6 PBRC05_P9 MAGE-A1 37% XXXXXXXXXXXXXXXXXXXX 3 3PBRC05_P10 MAGE-C2 21% XXXXXXXXXXXXXXXXXXXX 3 3 PBRC05_P11 MAGE-A12 13%XXXXXXXXXXXXXXXXXXXX 3 4 PBRC05_P12 SSX-2  6% XXXXXXXXXXXXXXXXXXXX 3 1PBRC05_P13 MORC ND XXXXXXXXXXXXXXXXXXXX 3 4 PBRC05_P14 MAGE-B1 NDXXXXXXXXXXXXXXXXXXXX 3 3 Note: Bold red means CD8 PEPI, Underline meansbest binding CD4 allele.

T cell responses were measured cells in peripheral mononuclear cells 2weeks after the 1^(st) vaccination with the mix of peptides PBRC05_P1,PBRC05_P2, PBRC05_P3, PBRC05_P4, PBRC05_P5, PBRC05_P6, PBRC05_P7.

TABLE 31 Antigen specific T cell responses: Number of spots/300,000 PBMCAntigen Stimulant Exp1 Exp2 Average SPAG9 PBRC05_P1 2 1 1.5 AKAP4PBRC05_P2 11 4 7.5 MAGE-A11 PBRC05_P3 26 32 29 NY-SAR-35 PBRC05_P4 472497 484.5 FSIP1 PBRC05_P5 317 321 319 NY-BR-1 PBRC05_P6 8 12 10 MAGE-A9PBRC05_P7 23 27 25 None Negative Control (DMSO) 0 3 1.5

The results show that a single immunization with 7 peptides inducedpotent T cell responses against 3 out of the 7 peptides demonstratingpotent MAGE-AM1, NY-SAR-35, FSIP1 and MAGE-A9 specific T cell responses.There were weak responses against AKAP4 and NY-BR-1 and no responseagainst SPAG9.

Example 20—Personalised Immunotherapy Composition for Treatment ofPatient with Early sAge Metastatic Breast Cancer

HISTORY: In 2011 left breast sector excision due to neoplasm. Treatment:aromatase inhibitor and lumbar spine irradiation (osseal mets).

In 2017, before PIT vaccine treatment was administered, a metastaticlesion was observed on the ventral bow of the right 5^(th) rib and inthe right 3^(rd) rib. In the left breast recurrent malignancy has to beruled out. In the right breast a malignancy with metastatic rightaxillary lymph node may exist.

TABLE 32 Vaccine peptides for patient of Example 20 Patient's vaccineTarget Antigen MAXHLA MAXHLA peptides Antigen Expression 20mer peptideCD8 CD4 PBRC04_P1 SPAG9 88% XXXXXXXXXXXXXXXXXXXX 3 1 PBRC04_P2 AKAP4 85%XXXXXXXXXXXXXXXXXXXX 4 4 PBRC04_P3 BORIS 71% XXXXXXXXXXXXXXXXXXXX 3 2PBRC04_P4 MAGE-A11 59% XXXXXXXXXXXXXXXXXXXX 3 1 PBRC04_P6 NY-SAR-35 49%XXXXXXXXXXXXXXXXXXXX 3 5 PBRC04_P7 FSIP1 49% XXXXXXXXXXXXXXXXXXXX 3 6PBRC04_P8 NY-BR-1 47% XXXXXXXXXXXXXXXXXXXX 3 1 PBRC04_P10 LDHC 35%XXXXXXXXXXXXXXXXXXXX 3 5 PBRC04_P11 GATA-3 31% XXXXXXXXXXXXXXXXXXXX 3 1PBRC04_P13 Survivin 71% XXXXXXXXXXXXXXXXXXXX 3 2 PBRC04_P14 MAGE-C1 12%XXXXXXXXXXXXXXXXXXXX 3 8 PBRC04_P15 PRAME 55% XXXXXXXXXXXXXXXXXXXX 3 5

The patient obtained 2 cycles of PIT vaccine.

Example 21—Characterization of Toxicity—immunoBLAST

A method was developed for performing on any antigen to determine itspotential to induce toxic immune reaction, such as autoimmunity. Themethod is referred to herein as immunoBLAST.

PolyPEPI1018 contains six 30-mer polypeptides. Each polypeptide consistsof two 15-mer peptide fragments derived from antigens expressed in CRC.Neoepitopes might be generated in the joint region of the two 15-merpeptides and could induce undesired T cell responses against healthycells (autoimmunity). This was assesses using the the immunoBLASTmethodology.

A 16-mer peptide for each of the 30-mer components of PolyPEP1018 wasdesigned. Each 16-mer contains 8 amino acids from the end of the first15 residues of the 30-mer and 8 amino acids from the beginning of thesecond 15 residues of the 30-mer—thus precisely spanning the jointregion of the two 15-mers. These 16-mers are then analysed to identifycross-reactive regions of local similarity with human sequences usingBLAST (blast.ncbi.nlm.nih.gov/Blast.cgi), which compares proteinsequences to sequence databases and calculates the statisticalsignificance of matches. 8-mers within the 16-mers were selected as theexamination length since that length represents the minimum lengthneeded for a peptide to form an epitope, and is the distance between theanchor points during HLA binding.

As shown in FIG. 16, the positions of amino acids in a polypeptide arenumbered. The start positions of potential 9-mer peptides that can bindto HLAs and form neoepitopes are the 8 amino acids in positions 8-15.The start positions of tumor antigen derived peptides harbored by the15-mers that can form the pharmaceutically active epitopes are 7+7=14amino acids at position 1-7 and 16-22. The ratio of possible neoepitopegenerating peptides is 36.4% (8/22).

The PEPI3+ Test was used to identify neoepitopes and neoPEPI among the9-mer epitopes in the joint region. The risk of PolyPEPI1018 inducingunwanted T cell responses was assessed in the 433 subjects in the ModelPopulation by determining the proportion of subjects with PEPI3+ amongthe 9-mers in the joint region. The result of neoepitope/neoPEPIanalysis is summarized in Table 33. In the 433 subjects of the ModelPopulation, the average predicted epitope number that could be generatedby intracellular processing was 40.12. Neoepitopes were frequentlygenerated; 11.61 out of 40.12 (28.9%) epitopes are neoepitopes. Most ofthe peptides were able to be be identified as a neoepitope, but thenumber of subjects that present neoepitopes varied.

Epitopes harbored by PolyPEPII018 create an average of 5.21 PEPJ3+.These PEPIs can activate T cells in a subject. The amount of potentialneoPEPIs was much lower than neoepitopes (3.7%). There is a marginalpossibility that these neoPEPIs compete on T cell activation with PEPIsin some subjects. Importantly, the activated neoPEPI specific T cellshad no targets on healthy tissue.

TABLE 33 Identification of Potential Neoepitopes of PolyPEPi1018Epitope & PEPI3+ binding in 433 Subjects of the Model PopulationPolyPEPI Epitope Binding (1 x HLA) 1018 SEQ NeoEPPEPI3+binding (3 × HLA) Peptide Potential ID NeoEP I NeoPEPI ID:Neoepitope NO Sub# Sub% I count Sub# Sub% NeoPEPI count CRC-P1 QFPVSEGKS39 0 0.0% 7 0 0.0% 3 FPVSEGKSR 40 160 37.0% X 1 0.2% X PVSEGKSRY 41 15034.6% X 0 0.0% VSEGKSRYR 42 194 44.8% X 1 0.2% X SEGKSRYRA 43 113 26.1%X 0 0.0% EGKSRYRAQ 44 77 17.8% X 0 0.0% GKSRYRAQR 45 37 8.5% X 0 0.0%KSRYRAQRF 46 337 77.8% X 33 7.6% X CRC-P2 IELKHKART 47 32 7.4% X 7 00.0% 1 ELKHKARTA 48 63 14.5% X 0 0.0% LKHKARTAK 49 59 13.6% X 0 0.0%KHKARTAKK 50 166 38.3% X 1 0.2% X HKARTAKKV 51 0 0.0% 0 0.0% KARTAKKVR52 70 16.2% X 0 0.0% ARTAKKVRR 53 134 30.9% X 0 0.0% RTAKKVRRA 54 419.5% X 0 0.0% CRC-P3 EFSMQGLKD 55 0 0.0% 5 0 0.0% 1 FSMQGLKDE 56 18843.4% X 0 0.0% SMQGLKDE 57 138 31.9% X 0 0.0% K MQGLKDEK 58 16 3.7% X 00.0% V QGLKDEKVA 59 0 0.0% 0 0.0% GLKDEKVAE 60 0 0.0% 0 0.0% LKDEKVAEL61 186 43.0% X 3 0.7% X KDEKVAELV 62 51 11.8% X 0 0.0% CRC-P6 LLALMVGLK63 252 58.2% X 7 0 0.0% 1 LALMVGLKD 64 86 19.9% X 0 0.0% ALMVGLKD 65 6515.0% X 0 0.0% H LMVGLKDH 66 97 22.4% X 0 0.0% R MVGLKDHRI 67 67 15.5% X0 0.0% VGLKDHRIS 68 0 0.0% 0 0.0% GLKDHRIST 69 4 0.9% X 0 0.0% LKDHRISTF70 195 45.0% X 5 1.2% X CRC-P7 PALFKENRS 71 0 0.0% 5 0 0.0% 1 ALFKENRSG72 0 0.0% 0 0.0% LFKENRSGA 73 41 9.5% X 0 0.0% FKENRSGAV 74 114 26.3% X0 0.0% KENRSGAV 75 261 60.3% X 0 0.0% M ENRSGAVM 76 0 0.0% 0 0.0% SNRSGAVMS 77 227 52.4% X 0 0.0% E RSGAVMSER 78 197 45.5% X 2 0.5% XCRC-P8 AVLTKKFQK 79 181 41.8% X 7 0 0.0% 3 VLTKKFQKV 80 208 48.0% X 20.5% X LTKKFQKVN 81 0 0.0% 0 0.0% TKKFQKVNF 82 25 5.8% X 0 0.0%KKFQKVNFF 83 250 57.7% X 12 2.8% X KFQKVNFFF 84 273 63.0% X 23 5.3% XFQKVNFFFE 85 163 37.6% X 0 0.0% QKVNFFFER 86 110 25.4% X 0 0.0%Abbreviations: CRC = colorectal cancer; HLA = human leukocytic ant. gen;PEPI = personal epitope

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What is claimed is:
 1. A human subject-specific pharmaceuticalcomposition for treatment of a disease or disorder in a specific humansubject, comprising (a) at least two different polypeptides, each of theat least two different polypeptides being 10-50 amino acids in lengthand comprising a T cell epitope that binds at least three HLA class Imolecules of the subject and/or at least three HLA class II molecules ofthe subject, and wherein the T cell epitope of each of the at least twopolypeptides are different from each other; and (b) apharmaceutically-acceptable adjuvant.
 2. The human subject-specificpharmaceutical composition of claim 1, comprising at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, at least10, at least 11, or at least 12 different polypeptides.
 3. The humansubject-specific pharmaceutical composition of claim 1, comprising 3-40different polypeptides.
 4. The human subject-specific pharmaceuticalcomposition of claim 1, wherein the T cell epitope that binds at leastthree HLA class I molecules of the subject comprises 7 to 11 aminoacids, and/or the T cell epitope that binds at least three HLA class IImolecules comprises 13 to 17 amino acids.
 5. The human subject-specificpharmaceutical composition of claim 1, wherein the epitopes of the atleast two different polypeptides are from a single antigen.
 6. The humansubject-specific pharmaceutical composition of claim 1, wherein theepitopes of the at least two different polypeptides are from two or moredifferent antigens.
 7. The human subject-specific pharmaceuticalcomposition of claim 5, wherein the antigen is an antigen expressed by acancer cell, a neoantigen expressed by a cancer cell, acancer-associated antigen, a tumor-associated antigen, or an antigenexpressed by a target pathogenic organism, an antigen expressed by avirus, an antigen expressed by a bacterium, an antigen expressed by afungus, an antigen associated with an autoimmune disorder, or is anallergen.
 8. The human subject-specific pharmaceutical composition ofclaim 7, wherein the cancer cell is from the subject.
 9. The humansubject-specific pharmaceutical composition of claim 5, wherein theantigen is selected from the antigens listed in Tables 2 to
 7. 10. Thehuman subject-specific pharmaceutical composition of claim 1, whereinthe at least two different polypeptides further comprise up to 10 aminoacids flanking the T cell epitope that are part of a consecutivesequence flanking the epitope in a corresponding antigen.
 11. The humansubject-specific pharmaceutical composition of claim 1, wherein the atleast two different polypeptides further comprise up to 10 amino acidsflanking the T cell epitope that are not part of a consecutive sequenceflanking the epitope in a corresponding antigen.
 12. The humansubject-specific pharmaceutical composition of claim 1, wherein two ofthe at least two polypeptides are arranged end to end or overlapping ina joined polypeptide.
 13. The human subject-specific pharmaceuticalcomposition of claim 12, comprising two or more different joinedpolypeptides, wherein the two or more different joined polypeptidescomprise different epitopes from each other.
 14. The humansubject-specific pharmaceutical composition of claim 13, wherein thejoined polypeptides have been screened to eliminate substantially allneoepitopes that span a junction between the two polypeptides and that(i) corresponds to a fragment of a human polypeptide expressed inhealthy cells of the subject; (ii) is a T cell epitope capable ofbinding to at least two HLA class I molecules of the subject; or (iii)meets both requirements (i) and (ii).
 15. The human subject-specificpharmaceutical composition of claim 1, wherein the at least twopolypeptides do not comprise any amino acid sequences that (i)correspond to a fragment of a human polypeptide expressed in healthycells; or (ii) correspond to a fragment of a human polypeptide expressedin healthy cells and is a T cell epitope capable of binding to at leasttwo HLA class I molecules of the subject.
 16. The human subject-specificpharmaceutical composition of claim 1, further comprising apharmaceutically acceptable diluent, carrier, preservative, orcombination thereof.
 17. The human subject-specific pharmaceuticalcomposition of claim 1, wherein the adjuvant is selected from the groupconsisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, bacillusCalmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone,isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins(KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete),mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols,polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), andcombinations thereof.
 18. A kit comprising, one or more separatecontainers each container comprising: (i) one or more polypeptides being10-50 amino acids in length comprising an amino acid sequence that is aT cell epitope that binds at least three HLA class I molecules of thesubject and/or at least three HLA class II molecules of the subject; and(ii) a pharmaceutically acceptable adjuvant, diluent, carrier,preservative, or combination thereof.
 19. The kit of claim 18,comprising at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, or atleast 12 different polypeptides, wherein the amino acid sequence of theT cell epitope of each of the different polypeptides are different fromeach other.
 20. The kit of claim 18, further comprising a packageinsert.
 21. A human subject-specific pharmaceutical compositioncomprising: a nucleic acid molecule expressing two or more polypeptides,each polypeptide being 10-50 amino acids in length comprising a T cellepitope that binds at least three HLA class I molecules of the subjectand/or at least three HLA class II molecules of the subject, whereineach of the two or more polypeptides comprises a different T cellepitope, wherein the polypeptides do not comprise amino acid sequencesthat are adjacent to each other in a corresponding antigen.
 22. Thepharmaceutical composition of claim 21, wherein the nucleic acidmolecule expresses at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 11, or at least12 different polypeptides, each being 10-50 amino acids in lengthcomprising an amino acid sequence that is a T cell epitope that binds atleast three HLA class I molecules of the subject and/or at least threeHLA class II molecules of the subject, wherein the amino acid sequenceof the T cell epitope of each of the different polypeptides aredifferent from each other.
 23. A human subject-specific pharmaceuticalcomposition for treatment of a disease or disorder in a specific humansubject, comprising at least one different polypeptides, each of the atleast one different polypeptides comprising at least a first region anda second region, (i) the first region of 10-50 amino acids in lengthcomprising an amino acid sequence that is a T cell epitope that binds atleast three HLA class I molecules of the subject and/or at least threeHLA class II molecules of the subject, (ii) the second region of 10-50amino acids in length comprising an amino acid sequence that is a T cellepitope that binds at least three HLA class I molecules of the subjectand/or at least two HLA class II molecules of the subject, wherein theamino acid sequence of the T cell epitope of each of the first andsecond regions of each of the at least three different polypeptidescomprise different sequences.
 24. The human subject-specificpharmaceutical composition of claim 23, comprising at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, or at least 12 different polypeptides. 25.The human subject-specific pharmaceutical composition of claim 23,comprising 2-40 different polypeptides.
 26. The human subject-specificpharmaceutical composition of claim 23, wherein the T cell epitope thatbinds at least three HLA class I molecules of the subject comprises 7 to11 amino acids, and/or the T cell epitope that binds at least three HLAclass II molecules comprises 13 to 17 amino acids.
 27. The humansubject-specific pharmaceutical composition of claim 23, wherein theepitopes of the first and second regions are from a single antigen. 28.The human subject-specific pharmaceutical composition of claim 23,wherein the epitopes of the first and second regions are from two ormore different antigens.
 29. The human subject-specific pharmaceuticalcomposition of claim 27, wherein the antigen is an antigen expressed bya cancer cell, a neoantigen expressed by a cancer cell, acancer-associated antigen, a tumor-associated antigen, or an antigenexpressed by a target pathogenic organism, an antigen expressed by avirus, an antigen expressed by a bacterium, an antigen expressed by afungus, an antigen associated with an autoimmune disorder, or is anallergen.
 30. The human subject-specific pharmaceutical composition ofclaim 29, wherein the cancer cell is from the subject.
 31. The humansubject-specific pharmaceutical composition of claim 27, wherein theantigen is selected from the antigens listed in Tables 2 to
 7. 32. Thehuman subject-specific pharmaceutical composition of claim 23, whereinthe polypeptides have been screened to eliminate substantially allneoepitopes that span a junction between the two regions and that (i)corresponds to a fragment of a human polypeptide expressed in healthycells of the subject; (ii) is a T cell epitope capable of binding to atleast two HLA class I molecules of the subject; or (iii) meets bothrequirements (i) and (ii).
 33. The human subject-specific pharmaceuticalcomposition of claim 23, wherein the at least one polypeptides do notcomprise any amino acid sequences that (i) correspond to a fragment of ahuman polypeptide expressed in healthy cells; or (ii) correspond to afragment of a human polypeptide expressed in healthy cells and is a Tcell epitope capable of binding to at least two HLA class I molecules ofthe subject.
 34. The human subject-specific pharmaceutical compositionof claim 23, further comprising a pharmaceutically acceptable adjuvant,diluent, carrier, preservative, or combination thereof.
 35. The humansubject-specific pharmaceutical composition of claim 34, wherein theadjuvant is selected from the group consisting of Montanide ISA-51,QS-21, GM-CSF, cyclophosamide, bacillus Calmette-Guerin (BCG),corynbacterium parvum, levamisole, azimezone, isoprinisone,dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins (KLH), Freundsadjuvant (complete), Freunds adjuvant (incomplete), mineral gels,aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions,oil emulsions, dinitrophenol, diphtheria toxin (DT), and combinationsthereof.
 36. A method of preparing a human subject-specificpharmaceutical composition for use in a method of treatment of aspecific human subject, the method comprising: (j) selecting a fragmentof a polypeptide, which fragment has been identified as immunogenic forthe subject by a) determining whether the fragment comprises: 1) anamino acid sequence that is a T cell epitope capable of binding to atleast three HLA class I molecules of the subject; or 2) an amino acidsequence that is a T cell epitope capable of binding to at least threeHLA class II molecules of the subject; or 3) or meets both requirements(1) and (2); and b) identifying the sequence as a fragment of thepolypeptide that is immunogenic for the subject; (ii) selecting a firstsequence of up to 50 consecutive amino acids of the polypeptide, whichconsecutive amino acids comprise the amino acid sequence of the fragmentselected in step (i); and (iii) preparing a subject-specificpharmaceutical composition having as active ingredients one or morepolypeptides that together have all of the amino acid sequences selectedin the preceding steps.
 37. The method of claim 36, further comprisingprior to the preparing step repeating steps (i) to (ii) to select asecond amino acid sequence of up to 50 consecutive amino acids of thesame or a different polypeptide to the first amino acid sequence. 38.The method of claim 37, further comprising, further repeating prior tothe preparing step, steps (i) to (ii) one or more times to select one ormore additional amino acid sequences of up to 50 consecutive amino acidsof the same or different polypeptides to the first and second amino acidsequences.
 39. The method of claim 36, further comprising prior to thepreparing step selecting a longer fragment of the polypeptide if thefragment selected in step (i) is an HLA class I-binding epitope, whichlonger fragment comprises the fragment selected in step (i); and is a Tcell epitope capable of binding at least three HLA class II molecules ofthe subject.
 40. The method of claim 36, wherein each polypeptide eitherconsists of one of the selected amino acid sequences, or comprises orconsists of two or more of the selected amino acid sequences arrangedend to end or overlapping in a single joined polypeptide.
 41. The methodof claim 36, wherein any neoepitopes formed at the junction between anytwo of the selected amino acid sequences arranged end to end in a singlejoined polypeptide have been screened to eliminate substantially allpolypeptides comprising a neoepitope amino acid sequence that (i)corresponds to a fragment of a human polypeptide expressed in healthycells; (ii) is a T cell epitope capable of binding to at least two HLAclass I molecules of the subject; or (iii) meets both requirements (i)and (ii).
 42. The method of claim 36, wherein the one or morepolypeptides have been screened to eliminate polypeptides comprising anamino acid sequence that (i) corresponds to a fragment of a humanpolypeptide expressed in healthy cells; or (ii) corresponds to afragment of a human polypeptide expressed in healthy cells and is a Tcell epitope capable of binding to at least two HLA class I molecules ofthe subject.
 43. The method of claim 36, further comprising determiningHLA class I genotype and HLA class II genotype from a biological sampleof the subject prior to step (i).
 44. The method of claim 43, whereinthe determining HLA class I genotype and HLA class II genotype isperformed by sequence based typing (SBT) methods.
 45. The method ofclaim 43, wherein the determining HLA class I genotype and HLA class IIgenotype is performed by sequencing, next generation sequencing,sequence specific primer (SSP) methods, or sequence specificoligonucleotide (SSO) methods.
 46. The method of claim 43, wherein thebiological sample is blood, serum, plasma, saliva, buccal swab, urine,expiration, cell, or tissue.
 47. The method of claim 43, wherein thebiological sample is saliva or a buccal swab.
 48. A method of treating acancer in a specific human subject in need thereof comprising,administering to a specific human subject a pharmaceutical compositioncomprising at least one polypeptide, each of the at least onepolypeptide being 10-50 amino acids in length comprising a first aminoacid sequence that is a T cell epitope that binds at least three HLAclass I molecules of the subject and/or at least three HLA class IImolecules of the subject, wherein the T cell epitope of each of the atleast one polypeptide is from an antigen that is specific for thecancer.
 49. The method of claim 48, wherein the composition comprises atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, or at least 12 differentpolypeptides, wherein the amino acid sequence of the T cell epitope ofeach of the different polypeptides are different from each other, andare from one or more antigens that are expressed by a cancer cell fromthe subject.
 50. The method of claim 48, wherein the compositioncomprises 2-40 different polypeptides.
 51. The method of claim 48,wherein T cell epitope that binds at least three HLA class I moleculesof the subject comprises 7 to 11 amino acids, and/or the T cell epitopethat binds at least three HLA class II molecules comprises 13 to 17amino acids.
 52. The method of claim 48, wherein the compositioncomprises at least two different polypeptides and the epitopes of theamino acid sequences of the at least two different polypeptides are froma single antigen.
 53. The method of claim 48, wherein the compositioncomprises at least two different polypeptides and the epitopes of the atleast two different polypeptides are from two or more differentantigens.
 54. The method of claim 48, wherein the one or more antigen isa neoantigen expressed by a cancer cell, a cancer-associated antigen, ora tumor-associated antigen.
 55. The method of claim 48, wherein the oneor more antigen is selected from the antigens listed in Table
 2. 56. Themethod of claim 48, wherein the at least one different polypeptidesfurther comprise up to 10 amino acids flanking the T cell epitope thatare part of a consecutive sequence flanking the epitope in acorresponding antigen.
 57. The method of claim 48, wherein the at leastone different polypeptides further comprise up to 10 amino acidsflanking the T cell epitope that are not part of a consecutive sequenceflanking the epitope in a corresponding antigen.
 58. The method of claim48, wherein the composition comprises at least two differentpolypeptides and two of the polypeptides are arranged end to end oroverlapping in a joined polypeptide.
 59. The method of claim 58,comprising two or more different joined polypeptides, wherein the two ormore different joined polypeptides comprise different epitopes from eachother.
 60. The method of claim 59, wherein the joined polypeptides havebeen screened to eliminate substantially all neoepitopes that span ajunction between the two polypeptides and that (i) corresponds to afragment of a human polypeptide expressed in healthy cells of thesubject; (ii) is a T cell epitope capable of binding to at least two HLAclass I molecules of the subject; or (iii) meets both requirements (i)and (ii).
 61. The method of claim 48, wherein the at least onepolypeptide does not comprise any amino acid sequences that (i)correspond to a fragment of a human polypeptide expressed in healthycells; or (ii) correspond to a fragment of a human polypeptide expressedin healthy cells and is a T cell epitope capable of binding to at leasttwo HLA class I molecules of the subject.
 62. The method of claim 48,wherein the composition further comprises a pharmaceutically acceptableadjuvant, diluent, carrier, preservative, or combination thereof. 63.The method of claim 62, wherein the adjuvant is selected from the groupconsisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, bacillusCalmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone,isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins(KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete),mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols,polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), andcombinations thereof.
 64. The method of claim 48, further comprisingadministering a chemotherapeutic agent, a targeted therapy, radiationtherapy, a checkpoint inhibitor, another immunotherapy, or combinationthereof.
 65. A human subject-specific pharmaceutical composition fortreatment of a disease or disorder in a specific human subject,comprising (a) a polypeptide of 10-50 amino acids in length andcomprising a T cell epitope that binds at least three HLA class Imolecules of the subject and/or at least three HLA class II molecules ofthe subject; and (b) a pharmaceutically-acceptable adjuvant.
 66. Thehuman subject-specific pharmaceutical composition of claim 65,comprising at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, or atleast 12 different polypeptides, each of the different polypeptidesbeing 10-50 amino acids in length comprising a T cell epitope that bindsat least three HLA class I molecules of the subject and/or at leastthree HLA class II molecules of the subject, wherein the amino acidsequence of the T cell epitope of each of the different polypeptides aredifferent from each other.
 67. The human subject-specific pharmaceuticalcomposition of claim 66, comprising 2-40 different polypeptides.
 68. Thehuman subject-specific pharmaceutical composition of claim 65, whereinthe T cell epitope that binds at least three HLA class I molecules ofthe subject comprises 7 to 11 amino acids, and/or the T cell epitopethat binds at least three HLA class II molecules comprises 13 to 17amino acids.
 69. The human subject-specific pharmaceutical compositionof claim 66, comprising at least two different polypeptides, wherein theepitopes of the at least two different polypeptides are from a singleantigen.
 70. The human subject-specific pharmaceutical composition ofclaim 66, comprising at least two different polypeptides, wherein theepitopes of the at least two different polypeptides are from two or moredifferent antigens.
 71. The human subject-specific pharmaceuticalcomposition of claim 69, wherein the antigen is an antigen expressed bya cancer cell, a neoantigen expressed by a cancer cell, acancer-associated antigen, a tumor-associated antigen, or an antigenexpressed by a target pathogenic organism, an antigen expressed by avirus, an antigen expressed by a bacterium, an antigen expressed by afungus, an antigen associated with an autoimmune disorder, or is anallergen.
 72. The human subject-specific pharmaceutical composition ofclaim 71, wherein the cancer cell is from the subject.
 73. The humansubject-specific pharmaceutical composition of claim 69, wherein theantigen is selected from the antigens listed in Tables 2 to
 7. 74. Thehuman subject-specific pharmaceutical composition of claim 69,comprising at least two different polypeptides, wherein two of thepolypeptides are arranged end to end or overlapping in a joinedpolypeptide.
 75. The human subject-specific pharmaceutical compositionof claim 65, wherein the adjuvant is selected from the group consistingof Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, bacillusCalmette-Guerin (BCG), corynbacterium parvum, levamisole, azimezone,isoprinisone, dinitrochlorobenezene (DNCB), keyhole limpet hemocyanins(KLH), Freunds adjuvant (complete), Freunds adjuvant (incomplete),mineral gels, aluminum hydroxide (Alum), lysolecithin, pluronic polyols,polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT), andcombinations thereof.
 76. The human subject-specific pharmaceuticalcomposition of claim 65, comprising at least two different polypeptides,wherein two of the at least two polypeptides are arranged end to end oroverlapping in a joined polypeptide.
 77. The human subject-specificpharmaceutical composition of claim 76, comprising two or more differentjoined polypeptides, wherein the two or more different joinedpolypeptides comprise different epitopes from each other.
 78. The humansubject-specific pharmaceutical composition of claim 77, wherein thejoined polypeptides have been screened to eliminate substantially allneoepitopes that span a junction between the two polypeptides and that(i) corresponds to a fragment of a human polypeptide expressed inhealthy cells of the subject; (ii) is a T cell epitope capable ofbinding to at least two HLA class I molecules of the subject; or (iii)meets both requirements (i) and (ii).
 79. The human subject-specificpharmaceutical composition of claim 66, wherein the at least twopolypeptides do not comprise any amino acid sequences that (i)correspond to a fragment of a human polypeptide expressed in healthycells; or (ii) correspond to a fragment of a human polypeptide expressedin healthy cells and is a T cell epitope capable of binding to at leasttwo HLA class I molecules of the subject.
 80. A kit comprising: (a) afirst human subject-specific pharmaceutical composition comprising (i) afirst polypeptide of 10-50 amino acids in length and comprising a T cellepitope that binds at least three HLA class I molecules of the subjectand/or at least three HLA class II molecules of the subject; and (ii) apharmaceutically-acceptable adjuvant; (b) a second humansubject-specific pharmaceutical composition comprising (i) a secondpolypeptide of 10-50 amino acids in length and comprising a T cellepitope that binds at least three HLA class I molecules of the subjectand/or at least three HLA class II molecules of the subject; and (ii) apharmaceutically-acceptable adjuvant, wherein the first and secondpolypeptides comprise different T cell epitopes.
 81. The kit of claim80, wherein the first composition and/or the second composition compriseone or more additional polypeptides, wherein each additional polypeptidebeing of 10-50 amino acids in length comprising an amino acid sequencethat is a T cell epitope that binds at least three HLA class I moleculesof the subject and/or at least three HLA class II molecules of thesubject, wherein the amino acid sequences comprise different T cellepitopes.